Chapter 1 - Cells: The Fundamental Units of Life
• A cell is the basic structural and functional unit of any living organism
The Flow of Genetic Information
• DNA synthesis —> RNA synthesis —> protein synthesis —> protein synthesis —> protein (amino acids)
• Different cells express different genes
Microscopes
• Light microscope requires a bright light focused on specimen by lenses in the condenser, the specimen must be carefully
prepped to allow light to pass through, and an appropriate set of lenses (objective and eyepiece) arranged to focus on image.
• Magnify cells up to 1000 times and resolves detail as small as 0.2 micro meters (a limitation imposed by wavelike nature of
light)
• Living cells can be viewed with straight-froward (bright-field) optics, phase contrast optics, and interference contrast optics,
exploiting differences in the way light travels through regions of the cell with differing refractive indexes
• Fluorescence microscopy uses fluorescent dyes to stain cells. The first filter of the illuminating light filters the light before it
reaches the specimen, passing only those wavelengths that excite the fluorescent dye. The second filter blocks out this light and
passes only those wavelengths emitted when the dye fluorescent. Dyed objects show up in bright color on a dark background.
• Confocal microscopy is a specialized fluorescence microscope that builds up an image by scanning specimen with a laser beam.
A series of optical sections at different depths allows a 3D image to be made. A conventional fluorescence microscope can give
blurry images due to the presence of fluorescent structures above and below the plane of focus.
• Transmission electron microscope (TEM) is similar to a light one, but it uses a beam of electrons instead of a beam of light and
magnetic coils to focus the beam instead of glass lenses. The specimen is placed in a vacuum and stained with electron-dense
heavy metals that locally absorb or scatter electrons, removing them from the beam as it passes through the specimen.
• Scanning electron microscopy (SEM) involves a specimen coated in thin film of a heavy metal is scanned by a beam of
electrons brought to a focus on the specimen by magnetic coils that act as lenses. The microscope creates 3D images.
Prokaryotic Cells
• organisms whose cells have a nucleus are eukaryotes; organisms whose cells do not have a nucleus are prokaryotes
• A prokaryotic cell includes Bacteria, the most common form of life on earth (prokaryotes also include archaea)
• Bacteria can be spherical, rod-shaped, or spiral cells
• Prokaryotes often have a tough protective coat, or cell wall, surrounding the plasma membrane, which encloses a single
compartment containing the cytoplasm and DNA
• Some aerobic or anaerobic; very diverse (most)
• Mitochondria (organelles that generate energy in eukaryotic cells) are thought to have evolved from aerobic bacteria that took
to living inside the anaerobic ancestors of todays eukaryotic cells
• Split in 2 domains - bacteria and archaea
• Archaea found in too hostile environments for other cells
• Eubacteria have cell walls of peptidoglycan, cannot survive in extreme environments, more standard energy production
• Archaebacteria can live in extreme environments, cell walls made of different matter, less standard energy production
• Both have no nucleus, no organelles, single-celled, prokaryotes, evolved from common ancestor
Eukaryotic Cells
• The nucleus is enclosed within 2 concentric membranes that form the nuclear envelope, containing molecules of DNA extremely long polymers that encode genetic information
• DNA becomes visible as individual chromosomes when they become more compact as a cell divides
• Mitochondria are found to be enclosed in 2 different membranes, with the inner membrane formed into folds that project into
the interior of the organelle. They are generators of chemical energy for the cell and harness energy from the oxidation of food
molecules such as sugars, to make ATP - the basic chemical fuel that powers cell’s activities
• Mitochondria consumes oxygen and releases CO2 in a process called cellular respiration
• chloroplasts are found only in cells of plants and algae (not in animals or fungi). In addition to 2 surrounding membranes, they
have internal stacks of membranes containing the green pigment chlorophyll. They carry out photosynthesis - trapping the
energy of the sunlight in chlorophyll molecules and using this energy to produce energy-rich sugar molecules, releasing oxygen
as a by-product
• Like mitochondria, chloroplasts contain their own DNA, reproduce by dividing in 2, and are thought to have evolved from
photosynthetic bacteria engulfed by an early eukaryotic cell
• Produces sugars which is oxidized in mitochondria to produce ATP
• The endoplasmic reticulum (ER) is where most cell-membrane components, as well as materials destined for export from the
cell, are made.
• Smooth: lipid synthesis, steroid hormone synthesis, detoxification, storage of calcium ions
• Rough: protein synthesis, translocation, folding, glycosylation, antigen processing
• the Golgi apparatus modifies and packages molecules made in the ER. Involved in protein modification through glycosylation,
completion of glycolipid and shingomyelin synthesis
• Lysosomes are small organelles in which intracellular digestion occurs, releasing nutrients from ingested food particles and
breaking down unwanted molecules for either recycling within the cell of exception from the cell
• Degradation, turnover of organelles (autophagy), antigen processing
• Endosomes involved in sorting of protein between endocytic and exocytic traffic, sorting of receptors and ligands, signaling
• Peroxisomes are membrane-enclosed vesicles that provide a safe environment for hydrogen peroxide to inactivate toxic
molecules; synthesis and degradation of hydrogen peroxide, oxidation of fatty acids, phosphorespiration in plants
• Endocytosis- portions at plasma membrane tuck inward and pinch off to form vesicles that carry material captured from the
external medium into the cell
• Exocytosis - vesicles from the inside of the cell fuse with plasma membrane and release contents into external medium
• Cytosol - part of the cytoplasm that is not contained within intracellular membranes
• Cytoskeleton - includes system of protein filaments composed of 3 major filament types
• Actin: the thinnest; abundant in all eukaryotic cells but occur in large numbers in muscle cells, where they serve as central part
of the machinery responsible for muscle contraction
• Microtubules: the thickest, hollow tubes, used in dividing cells
• Intermediate filaments: serve to strengthen the cell
• Motor proteins use the energy stored in molecules of ATP to trundle along those tracks and cables, carrying organelles and
proteins throughout the cytoplasm, and racing across the width of the cell in seconds
Possible Evolution of Eukaryotic Endomembrane Systems
• invagination of the plasma membrane —> pinching off would create a double membrane surrounding the nucleus - ER would
be continuous with this nuclear envelope
• Eukaryotic, bacterial, and archaean lineages diverged. Later eukaryotes acquired mitochondria, and following a subset of
eukaryotes acquired chloroplasts. Mitochondria are essentially the same in plants, animals, and fungi, and are thought to be
acquired before these lineages diverged
Endosymbiosis
• invagination of plasma membrane
• Organelles and membranes evolved through autophagy
• Pinching off would create double membrane surrounding nucleus, consistent with the fact that ER would be continuous with
the nuclear envelope
• Eukaryotic cytoskeleton and endomembrane system evolved through phagotrophy
• Recovery entailed the evolution of the nucleus, mitotic cycle
Phagotrophy
• predation and endomembrane formation
• Primordial predation: pray adhesion, secretion of digestive enzymes, protein import and proteasome digestion
• Actin-driven pseudopodia evolved and engulfed prey through accidental fusion \, leading to formation of phagosome
• After digestion, membrane is recycled, and trash excreted through exocytosis
• Problems: because DNA and ribosomes were membrane tethered, vesicular membrane cross-talk, recycling was needed
otherwise DNA and ribosomes would be excreted
• Endomembrane Stabilization
• Budding of coated vesicles evolved
• Protomembranes began to carry DNA, ribosomes, protein translocations
• Plasma membrane would be devoid of DNA/ribosomes due to evolution of compartmentalization
• Coated vesicles (CV) transport need for phagosome membrane recycling
• Proteasome becomes associated with protoER
• Endomembrane differentiation
• Divergence of vesicles coats —> divergence of ER, protoGolgi
• Retrograde vesicles transport
• Homotypic fusion of vesicles into protoGolgi to create Golgi cisternae
• Differentiation of vesicles sorting leads to formation of golgi proximal/distal cisternae
• Nuclear envelope/pore complex also formed from homotypic vesicle fusion
• more evidence by ER feeding back into immune cells to facilitate phagocytosis
Physiological Pressures Driving Organelle Formation
• the eukaryotic cytoskeleton and endomembrane system evolved through phagotrophy
• Phagocytosis internalized DNA-membrane attachments, unavoidably distrusting cell division; recovery entailed the evolution
of the nucleus and mitotic cycle
• Symbiogenetic origin of mitochondria soon followed the perfection of phagotrophy and intracellular digestion
• Anaerobic pre-eukaryotic cell engulfs bacterium (aerobic prokaryotic cell) —> mitochondria lumen remains isolated from the
endomembrane system by deriving a membrane from the eukaryotic cell
—> mitochondria having separate genome in early aerobic eukaryotic cells
• Photosynthetic bacterium engulfed by early eukaryotic cell —> eukaryotic cell capable of photosynthesis by containing
chloroplasts
Symbiogenetic Cell Enslavement
• the site of oxygen consumption within cells
• Generation of energy in the form of ATP via oxidative phosphorylation (photosynthesis in chloroplasts)
• Possesses their own DNA, similar to prokaryotic DNA
• Possesses their own ribosomes, similar to prokaryotic ribosomes
• Synthesize many, but not all, of their own proteins
• Mitochondria replicate by binary fission - similar to prokaryotic cell division
Tree of Life - Bill Martin 1998
• shows chimeric origin of eukaryotes, in which an archaeal host cell acquired bacterial endosymbionts
that evolved into mitochondria; and the later acquisition of chloroplasts in planta
Chapter 3 - Chemical Components of the Cell
• Four types of macromolecules joined by carbon bonds to make larger molecules:
• Sugars —> polysaccharides, glycogen, starch (plants)
• Fatty acids —> fats and membrane lipids
• Amino acids —> proteins
• Nucleotides —> nucleic acids
• Macromolecules are the most abundant organic molecules in cells
Sugars
• The simplest sugars - monosaccharides - have the general formula (CH2O)n
• Sugars and the larger molecules made from them are also called carbohydrates
• Monosaccharides can be linked by covalent bonds - called glycosidic bonds - to form larger carbohydrates. Two
monosaccharides linked together —> disaccharide
• Larger polymers range from oligosaccharides up to giant polysaccharides
• A bond is formed between an -OH group on one sugar and an -OH on another by condensation reaction, in which a molecule
of water is expelled as the bond is formed
• These bonds can be broken through hydrolysis, in which a water molecule is consumed
• Cells use polysaccharides composed of glucose units - glycogen in animals and starch in plants - as long-term stores of glucose,
held in reserve for energy production
• Sugar cellulose is used for mechanical support in cell walls, chitin in insect exoskeleton
• Glycoproteins and glycolipids found in cell membrane to protect surface and promote cell to cell adherence. Glycolipids are
composed of hydrophobic region with 2 hydrocarbon tails, and a polar region which contains one or more sugar residues and no
phosphate
• Cellulose: polymer of glucose linear
• Chitin: insect exoskeleton and fungal cell wall; linear polymer of GlcNAc
• Cellular roles: energy source (quick use = glucose; long-term storage = glycogen/starch), mechanical support (cellulose, chitin),
and glycoproteins and glycolipids for intracellular signaling/cell surface receptor interaction/adhesion/protein interactions
Fatty Acids
• 2 chemically distinct regions: one is a long hydrocarbon chain (hydrophobic and not very reactive
chemically), the other is a carboxyl (COOH) group which behaves as an acid (hydrophilic and reactive) —> amphipathic
• Almost all fatty acid molecules in a cell are covalently bonded to other molecules by the carboxylic acid group
• The hydrocarbon tail is saturated - having no carbon-carbon double bonds, containing maximum possible number of
hydrogens
• Some fatty acids have unsaturated tails, creating kinks that interfere with their ability to pack together
• Fatty acids serve as a concentrated food reserve in cells and can be broken down to produce about 6 times as much usable
energy as glucose
• Stored in the cytoplasm of many cells as fat droplets composed of triacylglycerol molecules (3 fatty acid chains covalently
joined to a glycerol)
• Lipids are insoluble in water but soluble in fat and organic solvents and contain long hydrocarbon chains (fatty acids) or
multiple linked aromatic rings (steroids)
• Lipid bilayer is the basis of all cell membranes composed of phospholipids which are constructed mainly from fatty acids and
glycerol. The glycerol is joined to 2 fatty acid chains, rather than to 3 as in triacylglycerol. The remaining -OH on the glycerol is
linked to a hydrophilic phosphate group, which is in turn attached to a small hydrophilic compound
Amino Acids
• are small organic molecules with one defining property: they all possess a carboxylic acid group and an amino group, both
linked to their alpha-carbon atom
Each amino acid also has a side chain attached to its alpha-carbon, distinguishing one amino acid from another
• Cells us amino acids to build proteins - polymers made of amino acids
• The covalent bond between 2 adjacent amino acids in a protein chain is a peptide bond; the chain being a polypeptide
• Peptide bonds formed by condensation reactions, linking amino acids
• Polypeptide always has an amino (NH2) group and one end - its N-terminus - and a carboxyl (COOH) group and its other end its C-terminus (structural polarity)
• Same 20 amino acids found in all proteins
• Like sugars, all amino acids (except glycine) exist as optical isomers in D- and L- forms, but only L-forms are ever found in
proteins
• General formula: NH2, H, R (side chain), and COOH all bonded to same alpha-carbon
• R commonly one of 20 different side chains
• At pH 7 both amino and carboxyl groups are ionized
• No rotation around C-N bond, but long chains of amino acids contain single bonds that akin for amino acid flexibility
• Cellular roles: protein synthesis, neurotransmitters (GABA/Glutamate), precursors to other molecules, metabolites (serotonin,
melatonin)
Nucleotides
• The subunits of DNA and RNA (nucleic acids)
• A nucleotide consists of a nitrogen containing base, a five-carbon sugar, and one or more phosphate groups
• Nucleotides area nucleosides that contain one or more phosphate groups attached to the sugar, and they come in 2 main
forms: those containing ribose = ribonucleotides, and those containing deoxyribose = deoxyribonucleotides
• The nitrogen-containing rings of all these molecules are bases
• Under acidic conditions they can each bind an H+ (proton) and thereby increase concentration of OH- ions in aqueous
solutions
• RNA: transcribes genetic code, translated genetic code (tRNA), structural (ribosomal RNA)
• The base is linked to the same carbon (C1) used in sugar-sugar bonds
• The bases are nitrogen containing ring compounds, either pyrimidines (C/T/U) or purines (A/G)
• The phosphates are normally joined to the C5 hydroxyl of the ribose or deoxyribose sugar (5’). The phosphate makes the
nucleotide negatively charged.
• Nucleotides are joined together by phosphodiester bonds between 5’ and 3’ carbon atoms of the sugar ring, via a phosphate
group, to form nucleic acids. The linear sequence of nucleotides in a nucleic acid chain is commonly abbreviated by a one-letter
code with the 5’ end chain at the left
• Phosphodiester bond: 5’ and 3’ carbon
• DNA and RNA synthesized from 5’—> 3’
• sugar/phosphate backbone
• Nucleotides carry chemical energy in their easily hydrolyzed phosphoanhydride bonds. The ribonucleotide adenosine
triphosphate (ATP) participates in transfer of energy in metabolic reactions.
The terminal phosphate group is frequently split off by hydrolysis. Transfer of this phosphate group to other molecules releases
energy
• Have fundamental role of storage and retrieval of biological information
• RNA = A,G,C,U; DNA = A,G,C,T
• DNA has more stable, hydrogen-bonded helices, acts as a long-term repository for hereditary information, while singlestranded RNA is usually a more transient carrier of molecular instructions
• ATP: phosphorylated nucleotide composed of adenosine and 3 phosphate groups, ribose sugar, 2’ position of the ribose
consists of a hydroxyl group, a ribonucleotide, serves as energy currency of the
cell dATP: one of the 2 purine nucleotides used to synthesize DNA, consists of deoxyribose sugar, 2’ position of the deoxyribose
consists of a hydrogen, a deoxyribonucleotide, serves as a precursor for DNA synthesis
• Nucleotides can combine with other groups to form coenzymes (CoA)
• Nucleotides can be used as signaling molecules in the cell (cyclic AMP)
• Cellular roles of nucleic acids: genetic code, energy carriers, co-enzymes, cell signaling
Making Proteins
• mRNA - the staring point of translation
• DNA transcribed into mRNA —> mRNA translated into amino acids (each codon responds to one
amino acid) —> amino acids form polypeptide chains —> polypeptide chains fold into proteins
Breaking Down and Building Cellular Components
Chapter 3 - pages 83-115
Four Types of Macromolecules
• Sugars —> polysaccharides, glycogen, and starch (in plants)
• Fatty acids —> fats and membrane lipids
• Amino acids —> proteins
• Nucleotides —> nucleic acids
Catabolic and Anabolic Pathways
• catabolic and anabolic pathways constitute the cells metabolism
• The measure of a systems disorder is called the entropy of the system, the greater the disorder the greater the entropy
• In the process of performing chemical reactions that generate order, some energy is lost in the form of heat
• It is the tight coupling of heat production to an increase in order that distinguishes the metabolism of the cell from the
wasteful burning of fuel in a fire
• Food molecules enter a catabolic pathway to produce energy and loss of heat to make building blocks for biosynthesis
• These forms of energy enter an anabolic pathway to create the many molecules that form the cell
Photosynthetic Organisms Use Sunlight to Synthesize Organic Molecules
• CO2 + H2O —> O2 + sugar
• Live on the energy stored in chemical bonds or organic molecules
• Photosynthesis converts the electromagnetic energy in sunlight into chemical-bond energy in cells
• 2 stages:
• 1 - energy from sunlight is captured and stored as chemical bond energy in specialized molecules called activated carriers (ATP
and NADH). All of the oxygen in the air that we breathe is generated by the splitting of water molecules during this first stage
• 2 - the activated carriers are used to help drive a carbon fixation process, in which sugars are manufactured from carbon
dioxide gas
Cells Obtain Energy by the Oxidation of Organic Molecules
• In both plants and animals, energy is extracted from food molecules by a process of gradual oxidation, or controlled burning
• Cellular respiration: a cell obtains energy from sugars or other organic molecules by allowing the carbon and hydrogen atoms
in these molecules to combine with oxygen (become oxidized) to produce CO2 and H2O
• Photosynthesis and cellular respiration are complementary processes
• Photosynthesis uses the energy of sunlight to produce sugars and other organic molecules from the carbon atoms of CO2 in
the atmosphere
• Cellular respiration uses O2 to oxidize food molecules, releasing the same carbon atoms in the form of CO2, back to the
atmosphere
• carbon atoms cycle continuously through the biosphere Oxidation and Reduction Involve Electron Transfers
• Oxidation: the addition of oxygen atoms to a molecule
• Occurs in any reaction in which electrons are transferred from one atom to another
• Removal of electrons from an atom
• Reduction: addition of electrons to an atom
• Oxidation and reduction even applies when there is only a partial shift of electrons between atoms linked by a covalent bond
• LEO says GER
• A simple reduced carbon compound, such as methane, can be oxidized in a stepwise fashion by the successive replacement of
its covalently bonded hydrogen atoms with oxygen atoms (methane —> methanol)
• Carbon dioxide becomes progressively more reduced as its oxygen atoms are replaced with hydrogens
• Hydrogenation reactions - reductions
• Dehydrogenation reactions - oxidations
Free Energy and Catalysis
• catalysis: the acceleration of specific chemical reactions needed to sustain life builds highly ordered and energy-rich molecules
from small and simple ones (requires the input of energy)
• Enzymes lower the activation energy needed to initiate reactions in the cell
• Chemical Reactions Proceed in the Direction that Causes a Loss of Free Energy
• The spontaneous direction for any reaction is the direction that goes “downhill” - energetically favorable
• Paper + O2 —> smoke + ashes + heat + CO2 + H2O
• Enzymes Reduce the Energy Needed to Initiate Spontaneous Reactions
• A molecule requires a boost over an energy barrier (activation energy) before it can undergo a chemical reaction that moves it
to a lower energy (more stable) state
• Inside cells, the push over the energy barrier is aided by specialized proteins called enzymes
• Each enzyme binds tightly to one or two molecules, called substrates, and holds them in a way that greatly reduces the
activation energy needed to facilitate a specific chemical interaction between them
• Substrate binds to active site (scaffold to support and position) on binding sites and the catalytic site reduces the chemical
activation energy
• A substance that can lower the activation energy of a reaction is a catalyst - increases the rate of chemical reactions by
allowing a much larger proportion of the random collisions with surrounding molecules to kick the substrates over the energy
barrier
• Enzymes are highly selective and remain unchanged after participating in a reaction
• Enzymes change shape slightly as substrate binds
• Enzymes regulate metabolic pathways
• The Free-Energy Change for a Reaction Determines Whether it Can Occur
• The useful energy in a system = free energy (G)
• Energetically favorable reactions are those that create disorder by decreasing the free energy of the system = negative G
• Energetically unfavorable reactions create order and have a positive G (forming peptide bond between two amino acids)
• Cannot occur spontaneously, they take place only when they are coupled to a second reaction with a negative G that is large
enough to make the net G negative as well
• Enzymes create order by coupling energetically unfavorable reactions with energetically favorable ones
• Free energy measured in kcal/mol, measures the energy of a molecule which could in principle be used to do useful work at a
constant temperature, as in a living cell. Can also in joules
• Delta G Changes As a Reaction Proceeds Towards Equilibrium
• Because delta G changes as products accumulate and substrates are depleted, chemical reactions will generally proceed until
they reach a state of equilibrium - making the rates of the forward and reverse reactions equal (delta G = 0)
• The Standard Free Energy Change, Delta Go, Makes it Possible to Compare the Energetics of Different Reactions
• Delta G depends on the concentrations of reactants at T
• The standard free energy change of a reaction is independent of a concentration and depends only on the intrinsic characters
of the reacting molecules, based on their behavior under ideal conditions where the concentration of all the reactants are set to
the same fixed value of 1 mol/L
• Delta G can be calculated from standard free energy change if concentrations of reactants and products known
• Coupled Reactions
• Reactions can be coupled together if they share one or more intermediates
• The overall free energy change is the sum of the individual free energy change values
• A reaction that is unfavorable can be driven by a second, highly favorable reaction
• Sucrose is made in a reaction driven by the hydrolysis of ATP
• In sequential reactions, free energy changes are additive
• The Equilibrium Constant is Directly Proportional to Delta Go
• The ratio of substrate to product at equilibrium is called the reaction’s equilibrium constant, K
• K = concentration of the product over the concentration of the substrate at equilibrium
• In Complex Reactions, the Eq Constant Includes the Concentrations of All Reactants and Products
• When two reactants combine to form a single product, the equilibrium constant, K, = the concentration of the product over
the concentration of the reactants multiplied
• The Equilibrium Constant Indicates the Strength of Molecular Reactions
• Two molecules will bind to each other if the free-energy change for the interaction is negative, that is, if the free energy of the
resulting complex is lower than the sum of the free energies of the two partners when unbound
• K is commonly employed as the measure of the binding strength of a non-covalent interaction between 2 molecules
• The larger the K, the greater is the drop in free energy between the dissociated and associated states, and the more tightly the
two molecules will bind
• For Sequential Reactions, the Changes in Free Energy Are Additive
• Activated carriers can shuttle energy from one reaction site to another
• Thermal Motion Allows Enzymes to Find Their Substrates
• Rapid binding is possible because molecular motions are fast
• Because of heat energy molecules are constantly in motion and will explore cytosolic space by wandering randomly through a
process called diffusion
• Because proteins diffuse through the cytosol much more slowly than do small molecules, the rate at which an enzyme will
encounter its substrate depends on the concentration of the substrate
• An enzyme-substrate complex is formed and stabilized by the formation of multiple weak bonds that persists until random
thermal motion causes the molecules to dissociate again
• When 2 colliding molecules have poorly matching surfaces, few noncovalent bonds are formed and their total energy is
negligible compared to that of the thermal motion
• Two molecules dissociate as rapidly as they come together
• This prevents incorrect and unwanted associations
• Vmax and Km Measure Enzyme Performance
• To catalyze a reaction an enzyme binds to its substrate —> substrate undergoes reaction to form a product —> product
released and diffuses away
• At first, concentration of the enzyme-substrate complex rises in a linear fashion in direct proportion to substrate concentration
As more and more of the enzyme molecules become occupied, rate increase tapers off until at a very high concentration of
substrate it reaches a max value called Vmax
• This is when active sites of all enzyme molecules in the sample are fully occupied by the substrate
• Michaelis constant, Km, is defined as the concentration of substrate at which the enzyme works at half its maximum speed
• Small Km indicates strong binding, large Km indicates weak binding
• Enzyme will also lower the activation energy for the reverse reaction to the same degree
Activated Carriers and Biosynthesis
• energy is stored as chemical bond energy in a set of activated carriers, small organic molecules that contain one or more
energy-rich covalent bonds
• These molecules diffuse rapidly and carry their bond energy from the sites of energy generation to the sites where energy is
used for biosynthesis
• Activated carriers store energy in an easily exchangeable form, either as a readily transferable chemical group or as readily
transferable electrons
• ATP/NADH
• The Formation of an Activated Carrier is Coupled to an Energetically Favorable Reaction
• Energy capture is achieved by means of a coupled reaction, in which an energetically favorable
reaction is used to drive an energetically unfavorable one that produces an activated carrier or some other useful molecule
• Activated carriers carry energy formed from catabolism to anabolic reactions
• Such coupling required enzymes, which are fundamental to all of the energy transactions in a cell
• ATP carries high energy phosphates
• NADH/NADPH/FADH2 carries electrons and hydrogens
• Acetyl CoA carries acetyl group
• ATP is the Most Widely Used Activated Carrier
• Adenosine 5’ triphosphate
• ATP is synthesized in an energetically unfavorable phosphorylation reaction in which a phosphate group is added to ADP
• ATP gives up energy in an energetically favorable hydrolysis to ADP (addition of water) and an inorganic phosphate
• ATP hydrolysis often coupled to the transfer of the terminal phosphate in ATP to another molecule
• Any reaction involving transfer of a phosphate group to a molecule = phosphorylation (ex of condensation reactions)
• Energy Stored in ATP is Often Harnessed to Join Two Molecules Together
• Energy from ATP hydrolysis is used to convert to a higher-energy intermediate compound, which then reacts directly to form a
bond
• Forces energetically unfavorable reactions to occur by coupling with ATP hydrolysis in an enzyme-catalyzed reaction pathway
• NADH and NADPH Are Both Activated Carriers of Electrons
• Carry both 2 high energy electrons and hydrogen atom —> H- hydride ion
• When they pass their energy to a donor molecule, they become NAD+ and NADP+
• During a set of energy-yielding catabolic reactions, a hydride ion is removed from the substrate molecule and added to
nicotinamide ring of NAP+ or form NADPH (a typical redox; the substrate is oxidized and NADP+ is reduced)
• The hydride ion carried by NADPH is given up readily in a subsequent redox because the ring can achieve a more stable
arrangement of electrons without it
• This regenerates NADP+ by oxidizing NADPH and reducing the substrate
• NADPH and NADH Have Different Roles in Cells
• NADPH operates with enzymes that catalyze anabolic reactions
• NADH has a special role as an intermediate in the catabolic system of reactions that generate ATP through oxidation of food
molecules
Cells Make Use of Many Other Activated Carriers
• Other important reactions involve the transfers of acetyl, methyl, carboxyl, and glucose groups from activated carriers
• Acetyl CoA carries an acetyl group in a readily transferable linkage to add sequentially 2 carbon units in the biosynthesis of the
hydrocarbon tails in fatty acids
• Activated carriers usually generated in reactions coupled to ATP hydrolysis as shown in biotin
• Once biotin is carboxylated, biotin can transfer a carboxyl group to another molecule. Here, it transfers a carboxyl group to
pyruvate, producing oxaloacetate, a molecule needed in the citric acid cycle. Other enzymes use biotin to transfer
carboxylgroups to other acceptor molecules
• Synthesis of carboxylated biotin requires energy derived from ATP hydrolysis
• The Synthesis of Biological Polymers Requires an Energy Input
• Subunits (monomers) linked together by bonds formed during an enzyme catalyzed condensation reaction
• The reverse reaction - breakdown of polymers - occurs thru enzyme-catalyzed hydrolysis reactions (energetically favorable)
• Path of ATP hydrolysis can be altered so that it initially produces AMP and pyrophosphate, which it itself then hydrolyzed in
solution in a subsequent step making it very favorable (involved in synthesis of nucleic acids)
• Nucleoside monophosphate activated by sequential transfer of terminal phosphate groups from 2 ATP molecules. The high
energy intermediate formed - a nucleoside triphosphate - exists free in solution until it reacts with growing end of an RNA or
DNA chain with release of pyrophosphate.
Hydrolysis of the pyrophosphate is highly favorable and helps to drive the overall reaction in the direction of polynucleotide
synthesis
From DNA to Chromosomes
Chapter 5 - pages 171-193
The Structure of DNA
• chromosomes contain both DNA and protein
• A DNA Molecule Consists of 2 Complementary Chains of Nucleotides
• A molecule of DNA consists of 2 long polynucleotide chains composed of 4 types of nucleotide subunits and the 2 strands are
held together by hydrogen bonds between the base portions of the nucleotides
• Each nucleotide composed of a sugar phosphate covalently linked to a base
• Nucleotides covalently linked together into polynucleotide chains with a sugar phosphate backbone
• Polarities run antiparallel wound in a double helix
• Sugar is deoxyribose and the bases are either A, C, G, T
• One end of the strand has a hole (the 3’ hydroxyl) and the other a knob (the 5’ phosphate)
• 2 chains in DNA double helix held together by hydrogen-bonding between the bases on the different strands
• Each purine-pyrimidine pair is called a base pair, and this complementary base pairing enables the base pairs to be packed in
the energetically most favorable arrangement in the interior of the double helix
• Nucleotides linked together covalently by phosphodiester bonds through the 3’ hydroxyl group of one sugar and the the 5’
phosphate of the next
• 3’ end carries an unlinked -OH attached to the 3’ of the sugar ring and the 5’ end carries a free phosphate attached to 5’ of the
sugar ring
• The Structure of DNA Provides a Mechanism for Heredity
• The exact correspondence between the 4-letter nucleotide alphabet of DNA and the 20-letter amino acid alphabet of proteins
is called the genetic code
• Gene expression: the process by which the nucleotide sequence of a gene is transcribed into the nucleotide sequence of an
RNA molecule, which, in most cases, is then translated into the amino acid sequence of a protein
The Structure of Eukaryotic Chromosomes
• Very long double-stranded DNA molecules are packaged into chromosomes
• DNA is compacted in a way that allows it to remain accessible to all of the enzymes and other proteins that replicate it, repair
it, and control the expression of its genes
• Eukaryotic DNA is Packaged into Multiple Chromosomes
• 23 or 24 different types of chromosomes (Y for males)
• The complex of DNA and protein = chromatin
• Human cells contain 2 copies of each chromosome, one from mother and one from father
• The maternal and paternal chromosomes of a pair are called homologous chromosomes
• Chromosomes Contain Long Strings of Genes
• A gene is a segment of DNA that contains the instructions for making a particular protein or RNA molecule
• Most of the RNA molecules encoded by genes are subsequently used to produce a protein
• In each gene, only one of the 2 DNA strands encodes the information to make an RNA molecule
• Genome: the total genetic information carried by all the chromosomes in a cell or organism
• Genome includes coding and non-coding RNA
• Specialized DNA Sequences Are Required for DNA Replication and Chromosome Segregation
• Interphase is when chromosomes are duplicated and mitosis is when they are distributed into 2 daughter nuclei
• One type of nucleotide sequence acts as a replication origin, where replication of the DNA begins
• Another DNA sequence forms telomeres at each of the 2 ends of a chromosome. They contain repeated nucleotide sequences
that are required for the ends of the chromosomes to be replicated.
They also cap the ends of DNA, preventing them from being mistaken by the cell as broken DNA in need of repair
• Another specialized sequence called the centromere allows duplicated chromosomes to be separated during the M phase
• Once chromosomes condense, centromere attaches to mitotic spindle to each duplicated chromosome in a way that allows
once copy of each chromosome to be segregated to each daughter cell
• Interphase Chromosomes Are Not Randomly Distributed Within the Nucleus
• The nucleolus is where the parts of the different chromosomes carrying genes that encode ribosomal RNAs cluster together.
Here rRNA are synthesized and combine with proteins to form ribosomes, the cells protein synthesizing machines
• The DNA in Chromosomes Is Always Highly Condensed
• Nucleosomes Are the Basic Units of Eukaryotic Chromosome Structure
• The proteins that bind to DNA to form chromosomes are divided into 2 classes: histones and nonhistone chromosomal
proteins
• The complex of both classes of proteins with nuclear DNA = chromatin
• Histones are responsible for the first and most fundamental level of chromatin packaging, the nucleosome
• The string of DNA and each bead is a nucleosome core particle, which consists of DNA wound around a core of proteins
formed from histones
• An individual nucleosome core particle consists of a complex of 8 histones proteins and a stretch of double-stranded DNA
• The formation of nucleosomes converts a DNA molecule into chromatin thread that is approximately 1/3 length of the initial
piece of DNA, and provides the first level of DNA packingThe positive charges help the histones bind tightly to the negatively
charged sugar-phosphate backbone of DNA
• Each histone in the octamer also has a long, instructed N-terminal amino acid tail that extends out from the nucleosome core
particle that allows for several types of modifications that controls many aspects of chromatin structure
• Chromosome Packing Occurs on Multiple Levels
• The nucleosomes are further packed on top of one anther to generate a more compact structure, such as chromatin fiber
• This fiber depends on histone H1, which pulls adjacent nucleosomes together into a regular repeating array
• Chromatin fiber is folded into a series of loops that are further condensed to produce the interphase chromosome
• This compact string of loops thought to undergo a final level of packing to form mitotic chromosome
The Regulation of Chromosome Structure
• Changes in Nucleosome Structure Allow Access to DNA
• Chromatin-remodeling complexes are protein machines that use energy of ATP hydrolysis to change the position of the DNA
wrapped around the nucleosomes (inactivated during mitosis)
• The complexes attach to both the histone octamer and the DNA wrapped around it can locally alter the arrangement of
nucleosomes on the DNA, making it more or less accessible
• Acetyl, phosphate, or methyl groups can be added to and removed from the tails by enzymes that reside in the nucleus
• Some of these proteins promote chromatin condensation whereas others deco dense chromatin and facilitate access to DNA
• Like chromatin-remodeling complexes, the enzymes that modify histone tails are tightly regulated
• Histone modifying enzymes and chromatin remodeling complex work in concert
• Interphase Chromosomes Contain Both Condensed and More Extended Forms of Chromatin
• Interphase chromatin not uniformly packed
• Regions of the chromosome that contain genes being expressed more extended, while those that contain silent genes are
more condensed
• The most highly condensed form of interphase chromatin is called heterochromatin
• The rest of the interphase chromatin is called euchromatin
• Each type of chromatin structure is established and maintained by different sets of histone tail modifications that attract
distinct sets of nonhistone proteins
• Heterochromatin can spread because these histone tail modifications attract a set of heterochromatin-specific proteins,
including histone modifying enzymes, which then create the same histone tail modifications on adjacent nucleosomes
• Will continue to spread until it encounters a barrier DNA sequence that stops the propagation
• Because heterochromatin is so compact, genes that accidentally become packaged into heterochromatin usually faint to be
expressed
• When a cell divides, it generally passes on its histone modifications, chromatin structure, and gene expression patterns to 2
daughter cells
How Do Cells Replicate, Repair, and Recombine Their DNA?
Chapter 6: pages 197-219
DNA Replication
• DNA replication is semi-conservative
• Base Pairing Enables DNA Replication
• Each strand can serve as a template for the synthesis of a new complementary strand
• DNA replication produces 2 complete double helices
• Each parental strand serves as a template for one new strand —> each of the daughter DNA double helices ending up with one
of the original strands and one new
• DNA Synthesis Begins at Replication Origins
• The process of DNA synthesis is begun by initiator proteins that bind to specific DNA sequences called replication origins
• Although hydrogen bonds collectively make the DNA helix stable, individually each hydrogen bound is weak
• Once initiator protein binds to DNA at a replication origin and locally opens up the double helix, it attracts a group of proteins
to carry out DNA replication
• Two Replication Forks Form at Each Replication Origin
• DNA molecule in the process of being replicated contain y-shaped junctions called replication forks
• Two are formed at each origin and move away from the origin in opposite directions = bidirectional
• DNA Polymerase Synthesizes DNA Using a Parental Strand as Template
• The movement of a replication fork is driven by the action of the replication machine, at the heart of which is an enzyme called
DNA polymerase which catalyzes the addition of nucleotides to the 3’ end of a growing DNA strand
• The polymerization reaction involves the formation of a phosphodiester bond between the 3’ end of the growing DNA chain
and the 5’ phosphate group of the incoming nucleotide, which enters the reaction as deoxyribonucleoside triphosphate
• The energy of polymerization is provided by the incoming deoxyribonucleoside triphosphate itself - hydrolysis of one of its high
energy phosphate bonds, releasing pyrophosphate
• Pyrophosphate further hydrolyzed to inorganic phosphate which makes polymerization irreversible
• The Replication Fork is Asymmetrical
• At each replication fork, one new DNA strand is being made on a template that runs 3’-5’ and then other being made on a
template that runs 5’-3’
• All DNA polymerase add new subunits to the 3’ end of a DNA strand
• The DNA strand that appears to grow in the incorrect 3’-5’ direction is made discontinuously in successive separate small
pieces - with the DNA polymerase moving backwards with respect to the direction of the replication fork movement so that each
new fragment can be polymerized in the 5-3
• The resulting small DNA pieces = Okazaki fragments that later join to form strand
• The DNA strand made discontinuously is called the lagging strand and the other the leading strand
• DNA Polymerase is Self-Correcting
• The enzyme carefully monitors the base-pairing between each incoming nucleotide and template strand
• Only when match is correct does it catalyze the nucleotide addition reaction
• If it makes a rare mistake, it can correct the error through proofreading which takes place at the same time as DNA synthesis
and is carried out by a nuclease that cleaves the phosphodiester backbone
• Short Lengths of RNA Act as Primers for DNA Synthesis
• A different enzyme is used to begin a new polynucleotide strand by joining 2 nucleotides together without the need for a basepaired end. This enzyme makes a short length of a closely related type of nucleic acid - RNA - using the DNA strand as a
template
• This RNA is base-paired to the template strand and provides a base-paired 3’ end as a starting point for DNA polymerase. It
serves as a primer for DNA synthesis and the enzyme is a primase
• Primase is an example of RNA polymerase, an enzyme that synthesizes RNA using DNA as a template
• The RNA primer is synthesized on the DNA strand by complementary base-painting in exactly the same way as DNA, but U
replaces T
• For leading strand, RNA primer needed only to start replication at the replication origin
• For lagging strand, new primers are needed to keep polymerization going
• DNA polymerase will continue to elongate the fragment until it runs into next RNA primer
• A nuclease degrades the RNA primer, a DNA polymerase called repair polymerase replaces this RNA with DNA, and DNA ligase
joins the 5’ phosphate end of one DNA fragment to the adjacent 3’-hydroxyl end of the next
• Proteins at a Replication Fork Cooperate to Form a Replication Machine
• DNA helicases and single-strand DNA binding proteins unzip ahead of the replication fork so that the incoming nucleoside
triphosphates can from base pairs with each template strand
• The helicase sits at the very front of the replication machine where it uses the energy of ATP hydrolysis to propel itself forward
to pry apart the double helix
• Single-strand DNA binding proteins cling to the single stranded DNA exposed by the helicase, preventing the strands from reforming base pairs and keeping them in the elongated form so that they can serve as sufficient templates
• Cells use proteins called DNA topoisomerases to relieve the tension that causes the fork to get wound more tightly by
producing transient nicks in the DNA backbone, which temporarily releases the tension; they then reseal the nick before falling
off the DNA
• Another protein called a sliding clamp keeps DNA polymerase attached to the template, forming a ring around the newly
formed DNA helix and by tightly gripping the polymerase
• Assembly of the clamp requires the activity of the clamp ladder, which hydrolyzes ATP each time it locks a sliding clamp around
a newly formed DNA double helix
• Telomerase Replicates the Ends of Eukaryotic Chromosomes
• When a final RNA primer on the lagging strand is removed, there is no way to replace it
• We solve this problem with the long repetitive sequence of at the end of chromosomes called telomeres
• These telomeric sequences attract telomerase to the chromosome ends which uses an RNA template that is part of the
enzyme itself, extending the ends of the replicating lagging strand by adding multiple copies of the same short DNA sequence to
the template strand DNA Repair
• Most DNA damage is only temporary b/c it is immediately corrected by processes collectively called DNA repair
• DNA Damage Occurs Continually in Cells
• Purine bases become losers from DNA in the cells of your body by spontaneous reactions called depurination
• Deamination is spontaneous loss of an amino group from a cytosine in DNA to produce the base uracil
• UV damages DNA by promoting covalent linkage between 2 adjacent pyrimidine bases
• Cells Possess a Variety of Mechanisms for Repairing DNA
• Damaged DNA recognized and removed. Involves nucleases, which cleave the covalent bonds that join the damaged
nucleotides to the rest of the DNA strand, leaving a small gap on one strand
• A repair DNA polymerase binds to the 3’-hydroxyl end of the cut DNA strand and fills the gap by making a complementary copy
of the information stored in the undamaged strand
• The break in the sugar-phosphate backbone of the repaired strand is sealed by DNA ligase, the same enzyme that joined
Okazaki fragments
• A DNA Mismatch Repair System Removes Replication Errors That Escape Proofreading
• The cell has a backup system called mismatch repair that corrects errors
• Whenever a copying mistake is made, it leaves behind a mismatched nucleotide that becomes recognized by a complex of
mismatch repair proteins that then removes the portion of DNA that contains the error and resynthesizes it to restore the
correct sequence
• An inherited predisposition to certain cancers is caused by mutations in genes that encode mismatch repair proteins
• Double Stranded DNA Breaks Require Different Strategy to Repair
• First strategy involves rapidly sticking the broken ends back together, before the fragments drift apart and become lost =
nonhomologous end joining
• Other is called homologous recombination
Homologous Recombination Can Flawlessly Repair DNA Double-Strand Breaks
• Involves finding an intact template to guide the repair
• Most often occurs shortly after a cells DNA has been replicated before cell division, when the duplicated helices are close to
each other
• To initiate repair, nuclease chews back at 5’ end of the 2 broken strands at the break. Then, one of the broken 3’ ends invades
the unbroken homologous DNA duplex and searches for the complementary
sequence through base-pairing. Once found, the invading strand is elongated by a repair DNA polymerase, using complementary
strand as a template. The newly repaired strand rejoins it original partner. Repair is then completed by additional DNA synthesis
at the 3’ ends of both strands of the broken double helix, followed by DNA ligation
• Failure to Repair DNA Damage Can Have Severe Consequences for a Cell or Organism
• A single nucleotide change causes sickle-cell anemia
• Inherited disease
• Marfan syndrome
• Disorder of connective tissue
• Myopia
• Overgrown bones and loose joints
• Long thin arms and legs
• Overgrown rib cage and spinal curvature
• Unchecked cell proliferation = cancer, giving rise to variant cells, accelerates the rate at which such somatic cell variants arise
From DNA to Protein: Gene Expression
Chapter 7: pages 223-255
From DNA to RNA
• central dogma: DNA to RNA to protein
• DNA —> RNA = transcription
• RNA —> protein = translation
• Many identical RNA copies can bear made from the same gene, and each RNA molecule can direct the synthesis of many
identical protein molecules
• Portions of DNA Sequence Are Transcribed into RNA
• RNA is a linear polymer made of four different nucleotide subunits, linked together by phosphodiester bonds
• Because an RNA chain is single-stranded it can fold into a variety of shapes
• The ability of RNA to fold into a complex 3D shape allows it to carry out various functions in cells, in addition to conveying info
between DNA and protein
• Differences from DNA: can fold more, uses rubonucleotides, U instead of T, single stranded
• Transcription Produces RNA That Is Complementary to One Strand of DNA
• Transcription begins by opening and unwinding DNA
• One of the two strands of DNA then acts as a template for the synthesis of RNA
• Ribonucleotides are added one by one to the growing RNA chain by complementary base pairing
• Incoming nucleotide covalently linked to the growing RNA chain by RNA polymerase —> RNA transcript
• Unlike newly formed DNA strand, the RNA strand does not remain hydrogen bonded to the DNA template strand
• Because RNAs are copied from only a limited region of DNA, RNA molecules are much shorter than DNA molecules
• RNA is made from 5’-3’ while DNA is 3’-5’
• RNA polymerases catalyze the formation of phosphodiester bonds that link the nucleotides together and form the sugarphosphate backbone of the RNA chainRNA polymerases can start an RNA chain without a primer
• Mistakes in RNA transcripts have relatively minor consequences for a cell
• Cells Produce Various Types of RNA
• The RNA molecules encoded by genes that direct the synthesis of proteins are messenger RNAs (mRNAs)
• Ribosomal RNAs from the core of the ribosomes structure and catalyze protein synthesis
• MicroRNAs regulate gene expression
• Transfer RNAs serve as adaptors between mRNA and amino acids
• Other non coding RNAs are used in RNA splicing, gene regulation, telomere maintenance, more
• Gene expression refers to the process by which the information encoded in a DNA sequence is translated into a product that
has some effect on a cell or organism
• Signals in DNA Tell RNA Polymerase Where to Start and Finish Transcription
• RNA polymerase latches onto DNA only after it has encountered a gene region called a promoter, which contains a specific
sequence of nucleotides that lies immediately upstream of the starting point of RNA synthesis
• Once bound, opens up DNA and picks a template strand for complementary base pairing with incoming ribonucleotides
triphosphates, two of which are joined together by the polymerase to begin synthesis of the RNA chain
• Chain elongation continues until the enzyme encounters a second signal in the DNA, the terminator, where the polymerase
halts and releases the DNA template and RNA transcript
• In bacteria, it is a subunit of RNA polymerase, the sigma factor, that is primarily responsible for recognizing the promoter
sequence on the DNA
• Every promoter has a certain polarity: it contains two different nucleotide sequences upstream of the transcriptional start site
that position the RNA polymerase, ensuring that it binds to the promoter in only one orientation. Because the polymerase can
only synthesize RNA in the 5’-3’ direction once the enzyme is bound it mayst use the DNA strand oriented in the 3-5 direction as
its template
• Initiation of Eukaryotic Gene Transcription Is a Complex Process
• Eukaryotic cells have 3 RNA polymerases: RNA polymerase I, II, and III (bacteria have one). RNA polymerase I and III transcribe
the genes encoding transfer RNA, ribosomal RNA, and others that play structural and catalytic roles. RNA polymerase II
transcribes the majority of genes, including those that encode proteins and miRNAs
• Eukaryotic RNA polymerases require the assistance of a large set of accessory proteins while bacterial RNA polymerase is able
to initiate transcription on its own. General transcription factors must assemble at each eukaryotic promoter, along with
polymerase, before it can begin transcription
• A single gene can be controlled by a large variety of regulatory DNA sequences scattered along the DNA, and it enables
eukaryotes to engage in more complex forms of transcriptional regulation than do bacteria
• Eukaryotic transcription has to take into account the packaging of DNA into nucleosomes
• Eukaryotic RNA Polymerase Requires General Transcription Factors
• General transcription factors are accessory proteins that assemble on the promoter, where they position the RNA polymerase
and pull apart the DNA double helix to expose template strand, allowing polymerase to begin transcription (similar role to sigma
factors in bacteria)
• Assembly process begins with binding of general transcription factor TFIID to a short segment of DNA composed primarily of T
and A (TATA box), causing a local distortion of the helix to assist in assembly of other proteins at the promoter to form a
complete transcription initiation complex
• Liberating the RNA polymerase begins with addition of phosphate groups to the tail. This is initiated by TFIIH, which contains a
protein kinase as one of its subunits
• Once transcription begins, most of general transcription factors dissociate
• When RNA polymerase II finishes transcribing the gene, it is released from the DNA; the phosphates on its tail are stripped off
by protein phoshatases
• Only the dephosphorylated form of RNA polymerase II can initiate RNA synthesis Eukaryotic mRNAs Are Processes in the
Nucleus
• Transcription takes place in the nucleus but protein synthesis takes place on ribosomes in the cytoplasm
• Before transported to cytosol, RNA must go through RNA processing steps, which include capping, splicing, and
polyadenylation
• The enzymes responsible for RNA processing ride on phosphorylated tail of eukaryotic RNA polymerase II as it synthesizes RNA
molecule, and they process the transcript as it emerges from the polymerase
• RNA capping modifies the 5’ end of RNA transcript, the end synthesized first. Capped by addition of G bearing a methyl group,
which is attached to the 5’ end of the RNA in an unusual way
• Polyadenylation provides a newly transcribed mRNA with a special structure on 3’ end. 3’ end of mRNA is first trimmed by an
enzyme that cuts RNA chain at particular sequence. Transcript then finished off by a second enzyme that adds a series of
repeated A’s to the cut end —> poly-A tail
• These modifications increase stability of mRNA, facilitate its export to cytoplasm, and mark RNA and mRNA
• In Eukaryotes, Protein-Coding Genes Are Interrupted by Noncoding Sequences Called Introns
• Most protein-coding eukaryotic genes have their coding sequences interrupted by long, non coding, intervening sequences
called introns
• The scattered pieces of coding sequences (expressed sequences) or exons are usually shorter than introns
• Introns Are Removed From Pre-mRNAs by RNA Splicing
• After capping as as RNA polymerase II continues to transcribe the gene, RNA splicing begins, in which the introns are removed
from the newly synthesized RNA and the exons are stitched together
• Each intron contains a few short sequences that act as cues for its removal from the pre-mRNA
• RNA splicing carried out by RNA molecules rather than proteins (small nuclear RNAs or snRNAs are packaged with additional
proteins to form snRNPs)
• SnRNPS recognize splice site sequences through complementary base-pairing between their RNA components and the
sequences in the pre-mRNA
• Together these snRNPs form the core of the spliceosome, the assembly of RNA and proteins that carry out RNA splicing in the
nucleus
• The transcripts of many eukaryotic genes can be spliced in different ways, each of which can produce a distinct protein. Such
alternative splicing allows different proteins to be made from same gene
• Novel proteins appear to arise from the mixing and matching of different exons of preexisting genes
• Mature Eukaryotic mRNAs Are Exported from the Nucleus
• Only correctly processed mRNAs are exported and this selective transport is mediated by nuclear pore complexes, which
connect the nucleoplasm with the cytosol and act as gates that control which macromolecules can enter or leave
• mRNA Molecules Are Eventually Degraded in the Cytosol
• Each mRNA is eventually degraded into nucleotides by ribonucleases (RNAses) present in the cytosol
Control of Gene Expression - Chapter 8
262-281
An Overview of Gene Expression
• Gene expression is a complex process by which cells selectively direct the synthesis of the proteins and RNAs encoded in their
genome
• Cell differentiation arises because cells make and accumulate different sets of RNA and protein molecules: that is, they express
different gene
The Different Cell Types of a Multicellular Organism Contain the Same DNA
• Cells have the ability to change which genes they express without altering the nucleotide sequence of their DNA
• DNA in specialized cell types of multicellular organisms still contains the entire set of instructions needed to form a whole
organism
• Different Cell Types Produce Different Sets of Proteins
• A Cell Can Change the Expression of Its Genes in Response to External Signals
• The specialized cells in a multicellular organism are capable of altering their patterns of gene expression in response to
extracellular cues
• Gene Expression Can Be Regulated at Various Steps from DNA to RNA to Protein
• A cell can control the proteins it contains by:
• 1. Controlling when and how often a given genome is transcribed
• 2. Controlling how an RNA transcript is spliced or processed
• 3. Selecting which mRNAs are exported from the nucleus to the cytosol
• 4. Regulating how quickly certain mRNA molecules are degraded
• 5. Selecting which mRNAs are translated into protein by ribosomes
• 6. Regulating how rapidly specific proteins are destroyed after they have been made
How Transcriptional Switches Work
• Transcriptional regulators are proteins that bind to DNA and control gene transcription
• Transcription Regulators Bind to Regulatory DNA Sequences
• Promoter region of a gene binds the enzyme RNA polymerase and correctly orients the enzyme to begin its task of making an
RNA copy of the gene
• Promoters include a transcription initiation site, where RNA synthesis begins, plus a sequence of nucleotides that extends
upstream from the initiation site. This region contains sites required for the RNA polymerase to recognize the promoter.
• These sequences contain recognition sites for proteins that associate with active polymerase - sigma factor in bacteria or
general transcription factors in eukaryotes
• Nearly all genes have regulatory DNA sequences that are used to switch the gene on or off
• Regulatory DNA sequences must be recognized by proteins called transcription regulators. It is the binding of transcription
regulator to a regulatory DNA sequence that acts as the switch to control transcription.
• Proteins that recognize a specific nucleotide sequence do so because the surface of the protein fits tightly against the surface
features of the DNA double helix in that region that vary depending on the nucleotide sequence
• Different DNA-binding proteins will recognize different nucleotide sequences
• Many transcription regulators bind to the DNA helix as dimers. Dimerization doubles the area of contact with the DNA,
increasing strength and specificity of the protein-DNA interaction
• Transcriptional Switches Allow Cells to Respond to Changes in Their Environment
• Bacteria regulate the expression of many of their genes according to the food sources that are available in the environment
• Coordinately transcribed clusters of an mRNA molecule are called operons - common in bacteria, rare in eukaryotes, where
genes are transcribed and regulated individually
• When tryp concentrations are low, operon is transcribed; resulting mRNA is translated to produce a set of biosynthetic
enzymes, which work in tandem to synthesize tryptophan
• When tryp abundant, the amino acid is imported into the cell and shuts down production of enzymes, which are no longer
needed.
• Within operons promoter is a short DNA sequences called the operator thats recognized by a transcription regulator. When
regulator binds to operator, it blocks access of RNA polymerase to the promoter, preventing transcription of operon and
production of tryp-producing enzymes.
• Transcription regulator is known as the tryptophan repressor - the repressor can bind to DNA only if it has also bound several
molecules of tryptophan The tryptophan repressor is an allosteric protein: the binding of tryp causes a change in its 3D
structure so that the protein can bind to the operator sequence. When conc. drops, repressor no longer binds to DNA, and tryp
operon is transcribed
• Repressors Turn Genes Off and Activators Turn Them On
• Tryptophan repressor is a transcriptional repressor protein: in its active form, it switches genes off (represses them)
• Transcriptional activator proteins switch genes on or activate them and are only marginally able to bind and position RNA
polymerase on their own. These promoters can be made fully functional by activator proteins that bind nearby and contact RNA
polymerase to help it initiate transcription
• Activator proteins have to interact with a second molecule ti be able to bind DNA
• Bacterial activator protein CAP has to bind cAMP before it can bind to DNA. Genes activated by CAP are switched on in
response to an increase in intracellular cAMP concentration, which rises
when glucose is no longer available
• As a result, CAP drives production of enzymes that allow bacterium to digest other sugars
• An Activator and a Repressor Control the Lac Operon
• Lac operon controlled by both Lac repressor and CAP activator and encodes proteins needed to import and digest lactose
• In absence of glucose, cAMP is made, which activates CAP to switch on genes that allow the cell to utilize alternative sources of
carbon (lactose)
• The Lac repressor shuts off the operon in the absence of lactose
• Operon is highly expressed only when 2 conditions met: glucose must be absent and lactose present
• Eukaryotic Transcription Regulators Control Gene Expression from a Distance
• The DNA sites to which eukaryotic gene activators bind are enhancers and work when bound either upstream or downstream
from the gene (even at long distances)
• The DNA between the enhancer and promoter loops out to allow eukaryotic activator proteins to influence directly events that
take place at the promoter. DNA thus acts as a tether, allowing a protein that is bound to an enhancer to interact with the
proteins in the vicinity of the promoter
• Additional proteins serve to link distantly bound transcription regulators to these proteins at the promoter; the most
important of these regulators is a large complex of proteins, the Mediator
• Aids in assembly of general transcription factors and RNA polymerase to form large transcription complex at the promoter
• Eukaryotic repressor proteins decrease transcription by preventing assembly of same protein complex
• Eukaryotic transcription regulators also attract proteins that modify chromatin structure and affect accessibility of the
promoter to the general transcription factors and RNA polymerase
• Eukaryotic Transcription Regulators Help Initiate Transcription by Recruiting Chromatin-Modifying Proteins
• Nucleosomes can inhibit initiation of transcription if positioned over a promoter, physically blocking the assembly of general
transcription factors or RNA polymerase on the promoter
• Gene activators can recruit chromatin-modifying proteins to promoters
• Recruitment of histone acetyltransferases promotes attachment of acetyl groups to selected lysine in the tail of histone
proteins, altering chromatin structure, allowing greater accessibility to underlying DNA. The acetyl groups themselves attract
proteins that promote transcription.
• Gene repressor proteins can modify chromatin in ways that reduce transcription initiation
• Many repressors attract histone deacetylases - enzymes that remove acetyl groups from histone tails, reversing positive
effects that acetylation has on transcription initiation The Molecular Mechanisms That Create Specialized Cell Types
• Changes in gene expression, which are often triggered by a transient signal, must be remembered by the cell - cell memory and is a prerequisite for creation of organized tissues and maintenance of stably differentiated cell types
• Eukaryotic Genes Are Controlled by Combinations of Transcription RegulatorsCombinatorial control refers to the way that
groups of transcription regulators work together to determine the expression a single gene
• A typical gene is controlled by dozens of transcription regulators. These help assemble chromatin-remodeling complexes,
histone-modifying enzymes, RNA polymerase, and general transcription factors via the multi protein Mediator complex
• The Expression of Different Genes Can Be Coordinated by a Single Protein
• As long as different genes contain regulatory DNA sequences that are recognized by the same
transcription regulator, they can be switched on or off together, as a coordinated unit
• Coordinated regulation in humans can be seen with the cortisol receptor protein. In order to bind to regulatory sites in DNA,
this transcription regulator must first form a complex with a molecule of cortisol. In response, livers cells increase expression of
genes, one of which encodes tyrosine aminotrasnferase. All these genes are regulated by binding of the cortisol-receptor
complex to a regulatory sequence in the DNA of each gene
• Combinatorial Control Can Also Generate Different Cell Types
• The ability to switch many genes on or off using limited number of transcription regulators is also one of the means by which
eukaryotic cells diversify into particular types of cells during embryonic development
• Genes encoding muscle-specific proteins are all switched on coordinately as the muscle cell differentiates
• A small number of key transcription regulators, expressed only in potential muscle cells, that coordinate muscle-specific gene
expression and are thus crucial for muscle-cell differentiation. This set of regulators activated the transcription of the genes that
code for muscle-specific proteins by binding to specific DNA sequences present in their regulatory regions.
• Some transcription regulators can convert one specialized cell type to another
• Cells can express many different genes, as dictated by combination of transcription regulators that each cell type produces
• Specialized Cell Types Can Be Experimentally Reprogrammed to Become Pluripotent Stem Cells
• One type of differentiated cell can be converted into another type by artificial expression of specific transcription regulators
• Transcription regulators can coax various differentiated cells to de-differentiate into pluripotent stem cells that are capable of
giving rise to all the specialized cell types in the body
• Using defined set of transcription regulators cells can be reprogrammed to become induced pluripotent stem (iPS) cells - cells
that look and behave like pluripotent ES cells derived from embryos
• The Formation of an Entire Organ Can be Triggered by a Single Transcription Regulator
• Action of a single transcription regulator can produce e a cascade of regulators that, working in combination, lead to formation
of an organized group of many different types of cells
• Artificially induced expression of Drosophila Ey gene in precursor cells of the leg triggers the misplaced development of an eye
on the fly’s leg
• Epigenetic Mechanisms Allow Differentiated Cells to Maintain Their Identity
• Some highly specialized cells, including skeletal muscle and neurons, never divide again once differentiated = terminally
differentiated
• For a proliferating cell to maintain its identity - a property called cell memory - the patterns of gene expression responsible for
that identity must be remembered and passed on to its daughter cells
• Positive feedback loop, where a master transcription regulator activates transcription of its own gene, in addition to that of
other cell-type-specific genes. Each time cell divides, regulator is distributed to both daughter cells, where it continues to
stimulate the positive feedback loop
• DNA methylation occurs on certain cytosine bases. This covalent modification turns genes off by attracting proteins that bind
to methylated cytosines and block gene transcription. DNA methylation patterns are passed onto progeny by the action of an
enzyme that copies the methylation pattern on the parent DNA strand to the daughter strand as its synthesized Enzymes
responsible for covalent modifications may bind to parental histones and confer the same modifications to the new histones
nearby, reestablishing pattern of chromatin structure found in the parent chromosome
• Because these cell-memory mechanisms transmit patterns of gene expression from parent to daughter cell without altering
the actual nucleotide sequence of the DNA, they are considered to be forms of epigenetic inheritance
Post-Transcriptional Controls
• Post-transcriptional controls operate after transcription has begun and plays a crucial role in regulating expression
• Alternative-RNA splicing allows different forms of a protein, encoded by the same gene, to be made in different tissues
• Each mRNA Controls Its Own Degradation and Translation
• mRNA’s lifetime is dictated by specific nucleotide sequences within the untranslated regions that lie both upstream and
downstream of the protein-coding sequence. These sequences often harbor binding sites for proteins that are involved in RNA
degradation
• Each mRNA possesses sequences that help control how often or how efficiently it will be translated into a protein, controlling
translation initiation
• Bacterial mRNAs contain short ribosome-binding sequence upstream of the AUG codon where translation begins. This
sequence forms base pairs with the RNA in the small ribosomal subunit, correctly positioning and initiating AUG codon within
the ribosome. By blocking or exposing this sequence, the bacterium can inhibit or promote translation of an mRNA.
• Eukaryotic mRNAs possess a 5’ cap that guides ribosome to first AUG. Repressor proteins can inhibit translation initiation by
binding to specific nucleotide sequences in the 5’ untranslated region, preventing ribosome from finding first AUG. When
conditions change, cell can inactivate repressor to initiate translation of mRNA.
Chapter 4 - Protein Structure and Function
Pages 121-161
• Proteins are the main building blocks from which cells are assembled, constituting most of the cell’s dry mass
• Enzymes catalyze covalent bond breaking or formation
• Structural proteins provide mechanical support to cells and tissues
• Transport proteins carry small molecules or ions
• Motor proteins generate movement in cells and tissues
• Storage proteins store amino acids or ions
• Signal proteins carry extracellular signals from cell to cell
• Receptor proteins detect signals and transmit them to the cell’s response machinery
• Gene regulatory proteins bind to DNA to switch genes on or off
• Special-purpose proteins are highly variable
The Shape and Structure of Proteins
• the position of each amino acid in the long string of amino acids forms a protein and determines its 3D shape, which is
stabilized by non covalent interactions between different parts of the molecule
• The Shape of a Protein Is Specified by Its Amino Acid Sequence
• A protein molecule is made from a long chain of amino acids held together by peptide bonds
• Proteins are referred to as polypeptides, and their amino acid chains are polypeptide chains
• Amino acids are present in a unique order called the amino acid sequence
• Each polypeptide chain consists of a backbone that is adorned with a variety of chemical side chains forming a polypeptide
backbone from a repeating sequence of the core atoms (-N-C-C-)
• Two ends of each amino acid are chem different: one with an amino group, forming N-terminus and other with a carboxyl,
forming the C-terminus
Projecting from the polypeptide backbone are the amino acid side chains not involved in forming peptide bonds. These give
each amino acid its unique properties: some non polar and hydrophobic and some negatively or positively charged, some can be
chemically reactive and so on
• Shape of each folded chains is constrained by many weak non covalent bonds within proteins - hydrogen bonds, electrostatic
interactions, and van der Waals attractions
• Hydrophobic interaction has a central role in determining shape of a protein, being forced together to minimize disruptive
effect on the hydrogen-bonded network of surrounding water molecules
• The non polar (hydrophobic) side chains cluster in interior of folded protein
• Polar side chains arrange near outside of folded protein, where they can form hydrogen bonds with water and other polar
molecules
• Proteins Fold into a Conformation of Lowest Energy
• Final folded structure, or conformation, is determined by energetic considerations: a protein folds into the shape in which its
free energy (G) is minimized
• Can be unfolded or denatured by treatment with solvents that disrupt noncovalent interactions holding the folded chain
together
• Removing denaturing solvent causes protein refolding spontaneously —> renaturation
• Conformation changes slightly when protein interacts with other molecules
• Misfolded proteins can damage cells and whole tissues —> neurodegenerative disorders
• A misfolded prion tends to form aggregates and can spread from cell to cell, converting properly folded versions to the
abnormal conformation, making prions “infectious”
• Protein folding in cells assisted by special proteins called chaperone proteins that bind to partly folded chains and help them to
fold along the most energetically favorable pathway
• “isolation chambers” can be formed in which single polypeptide chains can fold without risk of forming aggregates in crowded
cytoplasm
• Proteins Come in a Wide Variety of Complicated Shapes
• Proteins can be globular or fibrous, can form filaments, sheets, rings, or spheres
• Backbone model, ribbon model, wire model, space-filling model
• The α Helix and the β Sheet Are Common Folding Patterns
• These two patterns are common because they result from hydrogen bonds that form between the N-H and C=O groups in the
polypeptide backbone
• Amino acid side chains not involved in forming these hydrogen bonds, allowing alpha helices and beta sheets to be generated
by many different amino acid sequences
• Helices Form Readily in Biological Structures
• A helix is a regular structure that resembles a spiral staircase and generated by placing many similar subunits next to one
another, each in the same strictly repeated relationship to the one before
• Depending on twist, helix said to be either right-handed or left-handed
• α helix generated when a single polypeptide chain turns around itself to form a structurally rigid cylinder. A hydrogen
bondmade between every fourth amino acid, linking C=O of one peptide bond the N-H of another —> right-handed helix
• Short regions of α helix abundant in proteins in cell membranes. The portion that crosses the lipid bilayer usually forms and α
helix composed of amino acid with non polar side chains. The polypeptide backbone (hydrophilic) is hydrogen bonded to itself in
the α helix, and is shielded from hydrophobic lipid environment of membrane by its protruding non polar side chains
• Sometimes 2 or 3 α helices will wrap around one another —> coiled-coil that forms when α helices have most of their non
polar (hydrophobic) side chains on one side, so they can twist around each other with these side chains facing inward
• β Sheets Form Rigid Structures at the Core of Many Proteins
• β sheet made when hydrogen bonds form between segments of a polypeptide chain that lie side by side
• When neighboring segments run in same orientation = parallel β sheet; when opposite = antiparallel β sheet
They permit the formation of amyloid fibers - insoluble protein aggregates that include those associated with neurodegenerative
disorders such as Alzheimers and prion diseases
• Proteins Have Several Levels of Organization
• A proteins structure begins with its amino acid sequence = primary structure
• Next level of organization includes alpha helices and beta sheets that form within certain segments of the polypeptide chain =
secondary structure
• The full 3D conformation formed by an entire polypeptide chain - included alpha helices, beta sheets, random coils, loops, and
folds that form between N- and C- termini = tertiary structure
• If protein is formed as a complex of more than one polypeptide chain, then the complete structure = quaternary structure
• Protein domain = any segment of a polypeptide chain that can fold independently into a compact, stable structure
• Different domains often associated with different functions
• Many Proteins Also Contain Unstructured Regions
• Domains are usually connected by relatively unstructured lengths of polypeptide chain
• Regions of polypeptide chain lacking an definite structure, which continually bend and flex due to thermal buffeting are
intrinsically disordered sequences and often found as short stretches linking domains
• Being able to flex and bend, they can wrap around one or more target proteins, binding with both high specificity and low
affinity
• Provide flexibility while increasing the frequency of encounters between the domains
• Also ideal substrates for the addition of chemical groups that control the way many proteins behave
• Few of the Many Possible Polypeptide Chains Will be Useful
• For a polypeptide that is n amino acids long, 20^n different chains are possible
• Only a fraction of these are actually present due to maintaining stability
• Many potential proteins have been eliminated by natural selection through trial-and-error process that underlies evolution
• Proteins Can be Classified into Families
• Protein families group proteins in which each family member has an amino acid sequence and a 3D conformation that closely
resemble those of other family members
• Large Protein Molecules Often Contain More Than One Polypeptide Chain
• Same type of weak noncovalent bonds that enable a polypeptide chain to fold into a specific conformation also allow proteins
to bund to each other to produce larger structures
• Any region on a protein’s surface ghat interacts with another molecule through sets of noncovalent bonds = binding site
• The bonding of 2 folded polypeptide chains at this site —> larger protein, whose quaternary structure —> each polypeptide
chain being a subunit and each subunit may contain more than one domain
• Proteins Can Assemble into Filaments, Sheets, or Spheres
• A chain of identical proteins can form if the binding site on one protein molecule is complementary to another region on the
surface of another protein of the same type
• Since each protein is bound to its neighbor in an identical way, often —> helix the can profuse an extended protein filament
• Other sets of identical proteins associate to dorm tubes or spherical shells
• Some Types of Proteins Have Elongated Fibrous Shapes
• Globular proteins: polypeptide chain folds up into a compact shape like a ball with an irregular surface
• Proteins that have roles that require them to span a large distance generally have a simple, elongated 3D structure = fibrous
proteins
• Alpha-keratin is a dimer of 2 identical subunits, with the long alpha helices of each subunit forming a coiled-coil region that are
capped at either end by globular domains containing binding sites that allow them to assemble into roselike intermediate
filamentsFibrous proteins abundant outside cell, where form gel-like extracellular matrix that helps bind cells together to form
tissues (collagen)
• Extracellular Proteins Are Often Stabilized by Covalent Cross-Linkages
• To maintain structure, polypeptide chains are stabilized by covalent cross-linkages that either tie together two amino acids in
same polypeptide chain or join together many polypeptide chains in a large protein complex
• Most common covalent cross-links are sulfur-sulfur bonds = disulfide bonds (S-S); formed before protein is secreted by an
enzyme in ER that links together 2 -SH groups from cysteine side chains that are adjacent in the folded protein
• Reinforce protein’s most favored conformation
• disulfide bonds usually don’t form in the cytosol, where high concentration of reducing agents converts such bonds back to
cysteine -SH groups
How Proteins Work
• All Proteins Bind to Other Molecules
• The binding of a protein to other biological molecules always shows great specificity
• Any substance that is bound by a protein is a ligand for that protein
• The ability of a protein to bind selectively and with high affinity to a ligand is due to formation of a set of weak, noncovalent
interactions (H bonds, electrostatic attractions, van der Waals) plus favorable hydrophobic forces
• When poorly matching surfaces, few noncovalent interactions occur, and 2 molecules dissociate as rapidly as they come
together
• The region of a protein that associates with a ligand, known as its binding site, consists of a cavity in the protein surface
formed by a particular arrangement of amino acid side chains
• Atoms buried in interior of protein have no direct contact with ligand, but provide essential scaffold that gives surface its
contours and chemical properties
• There Are Billions of Different Antibodies, Each with a Different Binding Site
• Antibodies are immunoglobulin proteins produced by the immune system in response to foreign molecules, especially those
on the surface of an invading microorganism
• An antibody recognized target - antigen - with specificity
• Antibodies are Y-shaped with 2 identical antigen-binding sites, each of which is complementary to a small portion of the
surface of the antigen molecule
• Antigen-binding sites of antibodies are formed from several loops of polypeptide chain that protrude from the ends of a pair of
closely juxtaposed protein domains. The amino acid sequence in these loops can vary greatly without altering basic structure of
antibody
• Enzymes Are Powerful and Highly Specific Catalysts
• Enzymes are responsible for chemical transformations that occur in cells; they bind to one or more ligands, called substrates,
and convert them into chemically modified products; they act as catalysts that permit cells to make or break covalent bonds at
will
• Arranged into classes: hydrolase (hydrolytic cleavage), nuclease (break down nucleic acids by hydrolyzing bonds between
nucleotides), protease (breaks down proteins by hydrolyzing peptide bonds between amino acids); ligase (joins 2 molecules
together), isomerase (catalyzes rearrangement of bonds within a single molecule), polymerase (polymerization reactions),
kinase (adding phosphate groups to molecules), phosphatase (hydrolytic removal of a phosphate group), oxide-reductase,
ATPase
• Lysozyme Illustrates How an Enzyme Works
• Lysozyme is an enzyme that acts as a natural antibiotic in egg white, saliva, teats, nd other secretions; they sever the
polysaccharide chains that form the cell walls of bacteria
• The reaction catalyzed by lysozyme is a hydrolysis - adds water to a single bond between 2 adjacent sugar groups in the
polysaccharide chain —> breaking the bond (energetically favorable)
For a colliding water molecule to break a bond linking 2 sugars, the polysaccharide molecule has to be distorted in particular
shape - the transition state - in which the atoms around the bond have an altered geometry and electron distribution
• Lysozyme has a binding site on its surface (active site) that cradles the contours of its substrate molecule
• As soon as enzyme-substrate complex forms, the enzyme cuts the polysaccharide by catalyzing addition of a water moles ale
to one of its sugar-sugar bonds
• Lysozyme holds polysaccharide substrate in a way that one of the 2 sugars is distorted from its normal/stable conformation.
The bond to be broken is held close to 2 specific amino acids with acidic side chains located within active site of enzyme
• Conditions created in lysozyme active site that reduce activation energy necessary for hydrolysis to take place
• In reaction s with 2 or more substrates, the active site also acts as a template or mold that brings reactants together in proper
orientation for reaction to occur
• Binding to enzyme changes shape of substrate, bending bonds to drive the bond molecule toward a particular transition state
• Many Drugs Inhibit Enzymes
• Drugs work by blocking the activity of a particular enzyme
• Tightly Bound Small Molecules Add Extra Functions to Proteins
• By binding reversibly to dissolved oxygen gas through its iron atom, heme enables hemoglobin to pick up oxygen in the lungs
and realize it in tissues that need it
• When small molecules are attached to their protein, they become integral part of the protein molecule
• Proteins can be anchored to cell membranes through covalently attached lipid molecules
• Enzymes frequently have a small molecule or metal atom associated with their active site that assists with their catalytic
function
How Proteins Are Controlled
• Activities are regulated in a coordinated fashion so the cell can maintain itself in an optimal state, producing only those
molecules it required to thrive under the current conditions
• Regulation of protein activity:
• Cell controls amount of protein it contains by regulating expression of the gene that encodes that protein and by regulating
the rate at which the protein is degraded
• Cell controls enzymatic activities by confining sets of enzymes to particular sub cellular compartments
• Protein alters shape and therefore function
• The Catalytic Activities of Enzymes Are Often Regulated by Other Molecules
• Feedback inhibition is when an enzyme acting early in a reaction pathway is inhibited by a late product of that pathway
• It is negative regulation, preventing an enzyme from acting
• Positive regulation is when enzyme’s activity is stimulated by a regulatory molecule rather than being suppressed
• Allosteric Enzymes Have Two or More Binding Sites That Influence One Another
• Many enzymes have at least 2 different binding sites on surface: active site recognizes substrates and one or more sites that
recognize regulatory molecules
• Interaction between sites located in different regions on protein depends on conformational changes in the protein: binding of
a ligand to one of the sites causes a shift in structure which alters binding of a ligand to a second site
• Many protein molecules are allosteric - they can adopt 2 or more slightly different confrontations, and their activity can be
regulated by a shift from one to another
• Phosphorylation Can Control Protein Activity by Causing a Conformational Change
• Eukaryotic cells attach a phosphate group covalently to one or more of the protein’s amino acid side chains for regulation —>
major conformation change that affects the binding of ligands elsewhere on the protein surface, altering its activity
Removal of phosphate group returns protein to original conformation and activity
• This reversible protein phosphorylation controls activity of many types of proteins in eukaryotic cells
and occurs in response to signals that specify some change in a cell’s state
• Involves enzyme-catalyzed (protein kinase) transfer of terminal phosphate group of ATP to the hydroxyl group on side chain of
the protein
• Reverse reaction - removal of phosphate group - is catalyzed by protein phosphatase
• Phosphorylation can either stimulate protein activity or inhibit it
• Covalent Modifications Also Control the Location and Interaction of Proteins
• Phosphorylation can create docking sites where other proteins can bind, promoting the assembly of proteins into larger
complexes
• Many proteins are modified by the addition of an acetyl group to a lysine side chain
• Each modifying group is enzymatically added or removed depending on the needs of the chill
• GTP-Binding Proteins Are Also Regulated by the Cyclic Gain and Loss of a Phosphate Group
• The phosphate is part of a guanine nucleotide - GTP - that is bound tightly to various types of GTP-binding proteins that act as
molecular switches: they are in active conformation when GTP is bound, but they can hydrolyze GTP to GDP, which releases a
phosphate and flips protein to inactive conformation
• Also reversible; regained by dissociation of the GDP, followed by binding of GTP
• ATP Hydrolysis Allows Motor Proteins to Produce Directed Movement in Cells
• Motor proteins generate the forces responsible for muscle contraction and cell movements
• Protein can only wander randomly back and forth to allow for reversible shape changes
• Irreversibility achieved by coupling one of the conformational changes to the hydrolysis of an ATP molecule bound to the
protein - which is why motor proteins are also ATPases
• Free energy released when ATP hydrolyzed, making it unlikely that protein will reverse shape change
• Proteins Often Form Large Complexes That Function as Protein Machines
• Each central process in a cell is catalyzed by a highly coordinated, linked set of many proteins
• In most such protein machines, the hydrolysis of bound nucleoside triphosphates (ATP or GTP) drives an ordered series of
conformational changes in some of the individual protein subunits, enabling the ensemble of proteins to move coordinately
• Appropriate enzymes positioned to carry out successive reactions in a series
How Proteins Are Studied
• Proteins Can be Purified from Cells or Tissues
• The first step in any purification is to break open the cells to release their contents —> cell homogenate or extract, followed by
an initial fractionation to separate out the class of molecule of interest (ex: soluble proteins)
• Then isolate desired protein by purifying the protein through a series of chromatography steps, which use different materials
to separate the individual components into fractions based on the properties of the protein - such as size, shape, or electrical
charge
• Fractions then examined to determine which contain protein of interest and then pooled and subjected to additional
chromatography until desired protein obtained in pure form
• Most efficient protein chromatography separates polypeptides on basis of ability to bind to a particular molecule - affinity
chromatography
• Can also be used to isolate proteins that interact physically with the protein being studied
• Purified protein of interest attached tightly to column matrix; proteins that bind to it will remain in column and can be
removed by changing composition of washing solution
• Proteins can also be separated by electrophoresis - mix of proteins loaded onto polymer gel and subjected to an electric field;
polypeptides then migrate through gel at different speeds depending on size and net charge
• Yields bands or spots visually by staining; each band containing a different protein
• Determining a Protein’s Structure Begins by Determining its Amino Acid Sequence Mass spectrometry determines exact mass
of every peptide fragment in a purified protein, which then allows protein to be identified from a database
• Peptides blasted with a laser that heats them, causing them to become electrically charged (ionized) and ejected in the form of
a gas
• Accelerated by an electric field, peptide ions fly toward detector; the time it takes them to arrive is related to their mass and
charge (larger it is more slowly it moves; more highly charged, the faster it moves) —> fingerprint that identifies the protein
• The only way to discover precise folding pattern of any protein is by using X-ray crystallography or NMR
• Genetic Engineering Techniques Permit the Large-Scale Production, Design, and Analysis of Almost Any Protein
• Can employ techniques to produce new proteins and enzymes with novel structures or perform unusual tasks
• The Relatedness of Proteins Aids the Prediction of Protein Structure and Function
• The majority belong to protein families that share specific “sequence patterns”- stretches of amino acids that fold into distinct
structural domains
Chapter 9 - How Genes and Genomes Evolve
Pages 289-320
Generating Genetic Variation
• several basic types of genetic change are crucial in evolution
• Mutation within a gene: existing gene modified by mutation that changes single nucleotide or deletes or duplicates one or
more nucleotides, altering the splicing of a genes transcript or changing the stability, activity, location, or interactions of its
encoded protein or RNA product
• Mutation within regulatory DNA: when a where a gene is expressed can be affected by a mutation in the stretches of DNA
sequence that regulate a gene’s activity
• Gene duplication: a gene, large segment of DNA, or whole genome can be duplicated —> set of closely related genes within a
single cell
• Exon shuffling: 2 or more genes can be broken and rejoined —> hybrid gene with DNA segments belonging to separate genes
• Mobile genetic elements: specialized DNA sequences that can move from one chromosomal location to another can alter
activity or regulation of gene
• Horizontal gene transfer: piece of DNA transferred from the genome of one cell to that of another (rare in eukaryotes,
common in bacteria)
• In Sexually Reproducing Organisms, Only Changes to the Germ Line Are Passed On To Progeny
• Only the specialized reproductive cells - germ cells - carry a copy of its genome to next generation of organisms
• All the other cells of the body - somatic cells - die w/o leaving evolutionary descendants
• For mutation to be passed on to next generation, it must alter the germ line - the cell lineage that gives rise to germ cells
• Point Mutations Are Caused by Failures of the Normal Mechanisms for Copying and Repairing DNA
• Changes that affect a single nucleotide pair are point mutations typically arising from rare errors in DNA replication or repair
• Can destroy a gene’s activity or very rarely improve it; more often have no effect on organisms appearance, viability, or ability
to reproduce
• In cases where they occur within an exon, neutral mutations can change the third position of a codon such that the amino acid
it specifies is unchanged
• Point Mutations Can Change the Regulation of a GeneMutations in regulatory DNA more difficult to recognize because they
don’t affect protein sequence and can be located some distance from the coding sequence of the gene
• Examples include malaria and lactose
• DNA Duplications Give Rise to Families of Related Genes
• Gene duplication most important mechanism for generating new genes from old ones
• Once gene duplicated, each of the 2 copies is free to accumulate mutations that might allow it to perform a slightly different
function - as long as original activity of gene is not lost
• By repeated rounds of gene duplication and divergence over millions of years, one gene can give rise to a while family of
genes, each with a specialized function
• Many gene duplications believed to be generated by homologous recombination - mechanism for mending a broken double
helix; it allows an intact chromosome to be used as a template to repair a damaged sequence on its homolog
• Rarely, a recombination event can occur between a pair of shorter DNA sequences - identical or very similar - that fall on either
side of the gene. If not aligned properly, a lopsided exchange of genetic info can occur (unequal crossover) —> one chromosome
that has an extra copy of the gene and another with no copy
• Subsequent unequal crossovers can really add extra copies to the duplicated set
• The Evolution of the Globin Gene Family Shows How Gene Duplication and Divergence Can Produce New Proteins
• Gene duplications followed by mutation are thought to have given rise to 2 slightly different globin genes, one encoding alpha
globin, the other encoding beta globin
• The beta-like globing gene that is expressed in the fetus developed from duplication and divergence —> fetal hemoglobin with
higher affinity for oxygen, helping transfer oxygen from mother to fetus
• Further rounds of duplication in both genes —> more members of these families. Each duplicated genes have been modified
by point mutations that affect properties of final hemoglobin molecule and by changes in regulatory DNA that determine when
and how strongly each gene is expressed
• Pseudogenes disabled by the accumulation of many mutations (inactivating them) shows that not every DNA duplication leads
to new functional gene
• Whole-Genome Duplications Have Shaped the Evolutionary History of Many Species
• Rather than single genes being duplicated in a piecemeal fashion, the whole vertebrate genome was long ago duplicated in
one fell swoop
• Large-scale duplications can happen if cell division fails to occur after a round of genome replication in germ line of individual
• Once accidental doubling of genome occurs in germ-line cell, it will be passed on to germ-line progeny cells in that individual
and to any offspring these cells may produce
• Novel Genes Can Be Created by Exon Shuffling
• Exon shuffling - exons from one gene being added to another; can facilitate evolution of new proteins
• Promoted by the same type of recombination that gives rise to gene duplications
• Recombination occurs within the introns that surround the exons. If the introns are from 2 different genes, this recombination
can —> hybrid gene that includes complete exons from both
• The Evolution of Genomes Has Been Profoundly Influenced by the Movement of Mobile Genetic Elements
• Mobile genetic elements = DNA sequences that can move from one chromosomal location to another
• Inserting mobile genetic element into coding sequence of a gene or into its regulatory region can cause “spontaneous”
mutations
• Can disrupt gene’s activity if they land directly within its coding sequence = insertion mutation (can destroy gene’s capacity to
encode a useful protein)
• Mobile genetic elements carry DNA sequences recognized by specific transcription regulators, and if inserted into a regulatory
DNA region (near a gene), the gene can be brought under the control of these transcription regulators, changing the gene’s
expression pattern
Mobile genetic elements provide opportunity for genomes rearrangements by serving as targets of homologous recombination
• Genes Can be Exchanged Between Organisms by Horizontal Gene Transfer
• Horizontal gene transfer occurs when genes and other portions of genomes exchanged between individuals of different
species; exchanging DNA by conjugation Reconstructing Life’s Family Tree
• homologous genes - those that are similar in nucleotide sequence because of their common ancestry
• Genetic Changes That Provide a Selective Advantage Are Likely to Be Preserved
• Mutations that are selectively neutral may or may not be passed on
• Mutations that are deleterious will be lost
• Deleterious alterations in a gene that codes for an essential protein or RNA molecule cannot be lost, being highly conserved proteins they encode are very similar from organism to organism
• They encode crucial proteins such as DNA and RNA polymerases
• Closely Related Organisms Have Genomes That Are Similar in Organization As Well As Sequence
• Phylogenic tree - a diagram that depicts evolutionary relationships among a group of organisms
• Human chromosome 2 arose from fusion of 2 chromosomes that remain separate in chimp, gorilla, and orangutan
• Functionally Important Genome Regions Show Up As Islands of Conserved DNA Sequence
• Large-scale organization of human and mouse genomes has been scrambled by many chromosome breakages and
recombinations
• Conserved synteny - regions where corresponding genes are strung together in the same order in both species
• Purifying selection - elimination of individuals carrying mutations that interfere with important functions allows for us to see
regions where changes are n to tolerated
• Genome Comparisons Show That Vertebrate Genomes Gain and Lose DNA Rapidly
• The size differences among modern vertebrate genomes accounted for by small blocks of sequence being lost from and added
to genomes at rapid rate
• Sequence Conservation Allows Us to Trace Even the Most Distant Evolutionary Relationships
• To construct ultimate phylogenic tree, focused on one particular gene conserved in all living species: the gene that codes for
ribosomal RNA (rRNA) of the small ribosomal subunit
• Prokaryotes comprise 2 distinct groups - bacteria and archaea
• Three domains: bacteria, archaea, and eukaryotes
Transposons and Viruses
• mobile genetic elements, jumping genes, are found in all cells. Their DNA sequences make up almost half of human genomes
but lack ability to leave the cell in which they reside
• Not the case for their relatives, the viruses
• Mobile Genetic Elements Encode the Components They Need for Movement
• Mobile genetic elements also called transposons
• Most common are the DNA-only transposons - move from one place to another as a piece of DNA, as opposed to an RNA
intermediate
• Each mobile genetic element typically encodes a specialized enzyme called a transposase, that mediates its movement. They
recognize and act on unique DNA sequences present on each mobile genetic element
• Mobile genetic elements occasionally rearrange the DNA sequences of the genome in which they are embedded
• If 2 MGE recognized by same transposase integrate into neighboring regions of same chromosome, the DNA between them
can be accidentally excised and inserted into a different gene or chromosome
• The Human Genome Contains Two Major Families of Transposable Sequences
• 1/2 human genome made up of mobile genetic elements Some elements have moved from place to place using cut-and-paste
mechanism, but most have moved not as DNA, but via an RNA intermediate. These retrotransposons appear to be unique to
eukaryotes
• L1 element is transcribed into RNA by a host cell’s RNA polymerase. A double-stranded DNA copy of this RNA is then made
using an enzyme called reverse transcriptase, a DNA polymerase than can use RNA as a template. L1 element encodes the
reverse transcriptase itself. The DNA copy of the element free to reintegrate into another site of the genome
• Another type of retrotransposon, the Alu sequence, do not encode own reverse transcriptase and depend on enzymes already
present in the cell to help them move
• Viruses Can Move Between Cells and Organisms
• Viruses are also mobile but can escape from cells and move to other cells and organisms
• They are genomes enclosed by a protective protein coat and must enter a cell and coopt its molecular machinery to express
their genes, make their proteins, and reproduce
• Viral reproduction often lethal to host cells; infect cell breaks open (lyses), releasing progeny viruses, which can infect
neighboring cells
• Most viruses that cause human disease have genomes made of double-stranded DNA or single-stranded RNA
• Amount of genetic material that can be packaged inside a viral protein shell is limited; viruses must hijack their host’s
biochemical machinery to reproduce themselves
• Retroviruses Reverse the Normal Flow of Genetic Information
• Retroviruses found only in eukaryotic cells and resemble retrotransposons
• DNA synthesized using RNA template using reverse transcriptase encoded by retroviral genome
• When single-stranded RNA genome of retrovirus enters a cell, the reverse transcriptase brought with it makes a
complementary DNA strand to form a DNA/RNA hybrid double helix. The RNA strand is removed and reverse transcriptase now
synthesizes a complementary DNA strand —> DNA double helix
• DNA is then inserted or integrated into a randomly selected site in host by virally encoded integrase enzyme
• Then, copying of the integrated viral DNA into RNA by a host-cell RNA polymerase, which produces large numbers of singlestranded RNAs identical to the original infecting genome. These viral RNAs are then translated by the host-cell ribosomes to
produce the viral shell proteins, the envelope proteins, and reverse transcriptase—all of which are assembled with the RNA
genome into new virus particles.
Examining the Human Genome
• The Nucleotide Sequences of Human Genomes Show How Our Genes Are Arranged
• Less than 2% codes for proteins; almost half made up of mobile genetic elements
• Most of DNA is in noncoding introns
• Each gene associated with regulatory DNA sequences that ensure gene is expressed at proper level, time, and place
• About 5% of human genome highly conserved when compared with other mammalian genomes
• Accelerated Changes in Conserved Genome Sequences Help Reveal What Makes Us Human
• Some DNA sequences that have been highly conserved in most mammalian species are found to have changed exceptionally
fast during last 6 million years of human evolution
• Such accelerated regions are thought to reflect functions that have been especially important in making us unique
• Exhibiting most rapid change was found to encode a short, non-protein-coding RNA that is produced in human cerebral cortex
at critical time during brain development
• Compared to Neanderthals, sudden spurt of changes in modern humans in regions that include genes involved in metabolism,
brain development, shape of skeleton, rib cage, and head
• Genetic overlap between Neanderthals and modern humans from Europe and Asia suggest that ancestors may have mated
with Neanderthals before outcompeting them
Genome Variation Contributes to Our Individuality - But How?
• Great deal of the genetic variation in present-day humans was inherited from our early human ancestors
• Most of genetic variation in human genome takes the form of single base changes called single-nucleotide polymorphisms
(SNPs) - points in the genome that differ in nucleotide sequence between one portion of the population and other
• Copy-number variations (CNVs) - long stretch of DNA has been gained or lost
• CA repeats make ideal markers for distinguishing DNA of individual humans
• Differences in numbers of short tandem repeats at different positions in genome are used to identify
individuals by DNA fingerprinting in crime scenes
Chapter 11 – Membrane Structure
- Plasma membrane: a protein-studded, fatty film so thin that it cannot be seen directly in the light microscope
- Separates and protects its chemical components from the outside environment
- Consists of a two-ply sheet of lipid molecules with inserted proteins
- PM serves as a barrier to prevent the contents of the cell from escaping and mixing with the surrounding medium
- To facilitate exchanges, membrane is penetrated by highly selective channels and transporters – proteins that allow specific
small molecules and ions to be imported and exported
- Other proteins in membrane act as sensors, or receptors, that enable the cell to receive information about changes in its
environment
- If pierces, it quickly reseals (spontaneously)
- Composed of lipids and proteins as a lipid bilayer serving as a permeability barrier to most water-soluble molecules
The Lipid Bilayer
- Membrane Lipids Form Bilayers in Water
o Each lipid has a hydrophilic head and a hydrophobic tail
o Most abundant lipids in cell membranes are phospholipids, which have phosphate-containing, hydrophilic head linked to a pair
of hydrophobic tails
o Phosphatidylcholine has the small molecule choline attached to a phosphate group as its hydrophilic head
o Molecules with both hydrophilic and hydrophobic parts are termed amphipathic
o Hydrophilic molecules dissolve readily in water because they have either charged groups or uncharged polar groups that can
form electrostatic attractions or hydrogen bonds with water molecules
o Hydrophobic molecules are insoluble in water because all of their atoms are uncharged and nonpolar, so can’t form favorable
interactions with water molecules phospholipids cage-like structure
o Cage-like structure is more highly ordered than rest of the water, so formation requires free energy. Energy cost is minimized
when hydrophobic molecules cluster together, limiting contacts with surrounding water molecules
Energetically most favorable
o The same forces that drive the amphipathic molecules to form a bilayer help to make the bilayer self-sealing. Molecules in
bilayer will spontaneously rearrange to eliminate the free edge.
o If tear is large, the sheet may begin to fold in on itself and break up into separate closed vesicles
o Amphipathic molecules such as phospholipids necessarily assemble into self-sealing containers that define closed
compartments
The Lipid Bilayer Is a Flexible Two-Dimensional Fluid
o Aqueous environment surrounding bilayer prevents membrane lipids from escaping, but nothing stops these molecules from
moving and changing places with one another within the plane of the bilayer behaves as 2D fluid
o Lipid bilayer is also flexible
o Pure phospholipids will spontaneously form closed spherical vesicles, called liposomes, when added to water (synthetic bilayer
for studying)
o In synthetic bilayers, rare for phospholipid molecules to tumble from one half of the bilayer, or monolayer, to the other (“flipflop”)
o As a result of random thermal motions, lipid molecules continuously exchange places with their neighbors in the same
monolayer
This exchange rapid lateral diffusion of lipid molecules within the plane of each monolayer
o Lipid molecules in cell membrane not only flex their hydrocarbon tails, but also rotate rapidly about their long axis
- The Fluidity of a Lipid Bilayer Depends on Its Composition
o The closer and more regular the packing of the tails, the more viscous and less fluid the bilayer will be
o 2 major properties of hydrocarbon tails affect how tightly they pack in the bilayer: their length and the number of double
bonds they contain
o Shorter chain reduces tendency of hydrocarbon tails to interact with one another, and therefore increases the fluidity of the
bilayer
o The chain that harbors a double bond does not contain the maximum number of hydrogen atoms that could be attached to its
carbon backbone = unsaturated with respect to H
o The hydrocarbon tail with no double bonds has a full complement of hydrogen atoms = saturated
o Each double bond creates a small kink in the tail, which makes it more difficult for the tails to pack against one another
This is why lipid bilayers that contain a large proportion of unsaturated hydrocarbon tails are more fluid than those with lower
proportions
o In bacteria and yeast, which adopt to varying temperatures, both lengths and unsaturation of tails in bilayer are constantly
adjusted to maintain the membrane at a relatively constant fluidity
At higher temps, cell makes membrane lipids with tails that are longer and that contain fewer double bonds
o Membrane fluidity in animal cells is modulated by the inclusion of the sterol cholesterol
Cholesterol molecules are short and rigid and fill spaces between neighboring phospholipid molecules left by the kinks in their
unsaturated hydrocarbon tails Cholesterol tends to stiffen the bilayer, making it less flexible and less permeable
o Fluidity enables membrane proteins to diffuse rapidly in the plane of the bilayer and to interact with one another (ex: cell
signaling) Permits membrane lipids and proteins to diffuse from sites where they are inserted into the bilayer after their
synthesis to other regions of the cell Ensures membrane molecules are distributed evenly between daughter cells when a cell
divides
Also allows membranes to fuse with one another and mix their molecules
- Membrane Assembly Begins in the ER
o New phospholipids manufactured by enzymes bound to cytosolic surface of ER
o Using free fatty acids as substrates, the enzymes deposit newly made phospholipids in the cytosolic half of the bilayer
The transfer of lipids from one monolayer to the other is catalyzed by enzymes called scramblases, which remove randomly
selected phospholipids from one half of the lipid bilayer and insert them in the other
Allows newly made phospholipids to be redistributed equally between each monolayer of the ER membrane
o Some will remain in ER and rest used to supply membrane to other compartments in cell
- Certain Phospholipids Are Confined to One Side of the Membrane
o Most cell membranes are asymmetrical
o The golgi membrane contains another family of phospholipid-handling enzyme, called flippases that remove specific
phospholipids from the side of the bilayer facing the exterior space and flip them into the monolayer that faces the cytosol
o Flippases initiate and maintain the asymmetric arrangement of phospholipids that is preserved as membranes bud from one
organelle and fuse with another – or with the PM
All cell membranes have distinct inside and outside faces
Cytosolic monolayer always faces cytosol, while noncytosolic monolayer is exposed to either the cell exterior or interior space
(lumen) of organelle
o Glycolipids show dramatic lopsided distribution in cell membrane. Located in PM and only in noncytosolic half of the bilayer
Sugar groups acquired in golgi apparatus face cell exterior, where they form part of a continuous coat of carbohydrate that
surrounds and protects animal cells
Enzymes oriented such that sugars are added only to lipid molecules in noncytosolic half
Remains trapped in this monolayer, as no flippases that transfer glycolipids to cytosolic side
Membrane Proteins
- Most membrane functions are carried out by membrane proteins
- Functions:
o Transporters: ex is Na+ pump that actively pumps Na+ out and K+ in
o Ion channels: ex is K+ leak channel that allows K+ ions to leave cells, thereby having a major influence on cell excitability
o Anchors: ex are integrins that link intracellular actin filaments to extracellular matrix proteins
o Receptors: ex is platelet-derived groups factor (PDGF) receptor that binds extracellular PDGF and generates intracellular
signals that cause cell to grow and divide
o Enzymes: ex is adenylyl cyclase that catalyzes the production of the small intracellular signaling molecule cyclic AMP in
response to extracellular signals
- Membrane Proteins Associate with the Lipid Bilayer in Different Ways
o 1. Transmembrane proteins extend through bilayer and are amphipathic, having both hydrophobic and hydrophilic regions
o 2. Other membrane proteins located almost entirely in cytosol and are associated with cytosolic half of lipid bilayer by an
amphipathic alpha helix exposed on surface of protein
o 3. Some lie entirely outside bilayer, on one side or other, attached to the membrane only by one or more covalently attached
lipid groups
o 4. Others are bound indirectly to one or the other face of the membrane, held in place by their interactions with other
membrane proteins
o Proteins directly attached to lipid bilayer are known as integral membrane proteins
o Remaining membrane proteins are peripheral membrane proteins and can be released from membrane by more gentle
extraction procedures that interfere with protein-protein interactions but leave bilayer intact
- A Polypeptide Chain Usually Crosses the Lipid Bilayer as an Alpha HelixPortions of a transmembrane protein located on either
side of lipid bilayer are connected by specialized membrane-spanning segments of the polypeptide chain
o These segments which run through the hydrophobic environment are composed largely of amino acids with hydrophobic side
chains
o The peptide bonds that join the successive amino acids in a protein are normally polar, making the polypeptide backbone
hydrophilic
o Atoms forming backbone are driven to form hydrogen bonds with one another
o Hydrogen bonding is maximized if the polypeptide chain forms a regular alpha helix
o Hydrophobic side chains exposed on outside of helix, where they contact the hydrophobic lipid tails, while atoms in the
polypeptide backbone form hydrogen bonds with one another on the inside of the helix
o In many transmembrane proteins, polypeptide chain crosses the membrane only once (single-pass; many are receptors for
extracellular signals)
o Others function as channels, forming aqueous pores across the lipid bilayer to allow small, water-soluble molecules to cross
the membrane
These consist of a series of alpha helices that cross bilayer number of times (multipass)
Amino acids arranged so hydrophobic side chains fall on one side of helix, while hydrophilic side chains are concentrated on
other side
In hydrophobic environment, alpha helices pack side by side in a ring, with hydrophobic side chains exposed to the lipids of
membrane and the hydrophilic side chains forming the lining of a hydrophilic pore through lipid bilayer
o Some can cross lipid bilayer as a beta sheet rolled into a cylinder
Amino acid side chains that face inside the barrel (line aqueous channel) are mostly hydrophilic, while those on outside, which
contact hydrophobic core are hydrophobic
- Membrane Proteins Can Be Solubilized in Detergents
o First step to separate membrane protein involves solubilizing the membrane with agents that destroy the lipid bilayer by
disrupting hydrophobic associations (use detergents)
o Detergents are small, amphipathic, lipid-like molecules with a single hydrophobic tail
o In water they tend to aggregate into small clusters called micelles, rather than forming a bilayer as do the phospholipids which
are more cylindrical
o When mixed, hydrophobic ends of detergent molecules interact with membrane-spanning hydrophobic regions of
transmembrane proteins, as well as hydrophobic tails of phospholipid molecules
Disrupts lipid bilayer and separates proteins from most phospholipids
Hydrophilic end cause interactions to bring membrane proteins into solution as protein-detergent complexes that can be
separated from one another
Detergent solubilizes the phospholipids
- We Know the Complete Structure of Relatively Few Membrane Proteins
o Since membrane proteins have to be purified in detergent micelles that are often heterogeneous in size, they are harder to
crystallize than soluble proteins that inhabit cell cytosol or extracellular fluids
o Bacteriorhodopsin is a small protein found in large amounts in PM of an archaean
o It acts as a membrane transport protein that pumps H+ out of cell and gets its energy from
sunlight (so contains single light-absorbing nonprotein molecule, called retinal, that gives the protein purple color)
o This small hydrophobic molecule is covalently attached to one of its 7 transmembrane alpha helices
o When absorbs photon of light, changes shape
transfer of one H+ from the retinal to the outside of the bacterium
Retinal regenerated by taking up a H+ from cytosol, returning protein to its original conformation
o In presence of sunlight, thousands pump H+ out of cell, making a concentration gradient of H+ across the PM. Cell uses this
proton gradient to store energy and convert it to ATP
- The Plasma Membrane Is Reinforced by the Underlying Cell Cortex
o Cell membranes strengthened and supported by framework of proteins, attached to membrane via transmembrane proteins
o PM of animal cells is stabilized by meshwork of fibrous proteins, called the cell cortex, that is attached to the underside of the
membrane
o Main component of the cortex of red blood cells is the dimeric spectrin, a long, thin, flexible rod that forms a meshwork that
provides support for PM and maintains cell’s biconcave shape
o Spectrin meshwork connected to membrane through intracellular attachment proteins that link spectrin to specific
transmembrane proteins
o Genetic abnormalities anemia (fewer red blood cells than normal/spherical not flattened)
o Other cells also need their cortex to allow them to selectively take up materials, to change their shape actively, and to move
(also restrain diffusion of proteins within the PM)
- A Cell Can Restrict the Movement of Its Membrane Proteins
o Lateral diffusion – membrane is a 2D fluid, many of its proteins, like its lipids, can move freely within the plane of the lipid
bilayer
o Cells have ways of confining particular proteins to localized areas within the bilayer membrane, creating functionally
specialized regions, or membrane domains, on cell or organelle surface
o PM proteins can be tethered to structures outside the cell or to relatively immobile structures inside the cell
o Cells can create barriers that restrict particular membrane components to one membrane domain
o Asymmetric distribution of membrane proteins maintained by a barrier formed along the line where the cell is sealed to
adjacent epithelial cells by a so-called tight junction
Specialized junctional proteins form continuous belt around the cell where the cell contacts its neighbors, creating a seal
between adjacent PMs
Membrane proteins cannot diffuse past the junction
- The Cell Surface Is Coated with Carbohydrate
o Some lipids in outer layer of PM have sugars covalently attached to them
o Same is true for most proteins in PM. They have short chains of sugars, called oligosaccharides, linked to them; they care
called glycoproteins
o Other proteins, the proteoglycans, have one or more long polysaccharide chains
o All carbohydrates on glycoproteins, proteoglycans, and glycolipids is located on the outside of the PM, where it forms a sugar
coating called the carbohydrate layer or glycocalyx
o This carbohydrate layer helps protect cell surface from mechanical damage
o As oligosaccharides and polysaccharides absorb water, they also give cell a slimy surface, which helps motile cells such as
white blood cells to squeeze through narrow spaces and prevents blood cells from sticking to one another or to the walls of
blood vessels
o Also have important role in cell-cell recognition and adhesion
o Proteins called lectins are specialized to bind to particular oligosaccharide side chains of glycoproteins and glycolipids (very
diverse
Chapter 12 - Transport Across Cell Membranes
Lecture 13
- A few molecules, such as CO2 and O2, can simply diffuse across the membrane
- Vast majority depends on membrane transport proteins that span the lipid bilayer, providing private passageways across
membrane
- Two main classes that mediate transfer: transporters and channels
- Transporters shift small organic molecules or inorganic ions from one side of the membrane to the other by changing shape
- Channels form tiny hydrophobic pores across the membrane through which such substances can pass by diffusion
- Most channels only permit passage of inorganic ions = ion channels
- Since ion channels are charged, their movements create a voltage across the membrane that enable nerve cells to
communicate
Principles of Transmembrane Transport
- Lipid Bilayers Are Impermeable to Ions and Most Uncharged Polar Molecules
o The rate at which a molecule will diffuse across lipid bilayer depends on the size and its solubility properties
o The smaller the molecule and more hydrophobic, or nonpolar, it is, the more rapidly it will diffuse
o Small nonpolar molecules (O2 and CO2) dissolve readily in lipid bilayers and rapidly diffuses
o Uncharged polar molecules (uneven distribution of charge) also diffuse readily if small enough. Water and ethanol cross faster
than glycerol. Larger uncharged polar molecules like glucose cross hardly at all.
o Lipid bilayers are highly impermeable to all charged molecules, including inorganic, no matter how small. Its charges and
strong electrical attraction to water inhibits entry into inner hydrocarbon phase of the bilayer.
- The Ion Concentrations Inside a Cell Are Very Different from Those Outside
o The most important inorganic ions for cells are Na+, K+, Ca2+, Cl-, H+
o Na+ is most plentiful cation outside cell; K+ most abundant inside
o High concentration of Na+ outside is balanced by extracellular Clo High concentration of K+ inside cell balanced by variety of organic and inorganic anions including nucleic acids, proteins, and
many cell metabolites
- Differences in the Concentration of Inorganic Ions Across a Cell Membrane Create a
Membrane Potential
o Tiny excesses of positive or negative charge concentrated in the neighborhood of the plasma membrane occurs. Such electrical
imbalances generate voltage difference across the membrane = membrane potential
o In steady-state conditions, the voltage difference across the cell membrane holds steady = resting membrane potential (but
not zero)
o Value expressed as negative number because interior of cell more negative than exterior
- Cells Contain Two Classes of Membrane Transport Proteins: Transporters and Channels
o Each type of cell membrane has its own characteristic set of transport proteins
o Most membrane transport proteins have polypeptide chains that transverse lipid bilayer multiple times (multipass
transmembrane proteins)
o Channels discriminate on basis of size and electric charge: when channel is open, any ion
or molecule that is small enough and carries appropriate charge can pass through
o A transporter transfers only those that fit into specific binding sites on the protein
- Solutes Cross Membranes by Either Passive or Active Transport
Direction of transport depends only on the relative concentrations of the solute on either side of the membrane
o Molecules spontaneously flow “downhill” – from high to low conc. = passive transport
o Passive transport characterized without expenditure of energy by transport protein
o All channels and many transporters act as conduits for passive transport
o The movement of a solute against its conc. gradient is active transport, and is carried out by special types of transporters
called pumps, which harness an energy source to power the transport process
- Both the Concentration Gradient and Membrane Potential Influence Passive Transport of Charged Solutes
o For uncharged molecule, direction of passive transport determined solely by conc. gradient
o For electrically charged molecules, the membrane potential tends to pull positively charged solutes into the cell and drive
negatively charged ones out
o A charged solute also tends to move down its conc. gradient
o Net force driving a charged solute across a cell membrane is a composite of 2 forces, one due to the conc. gradient and other
due to membrane potential
o Net driving force called the solute’s electrochemical gradient, determines direction that solute will flow across membrane by
passive transport
- Water Moves Passively Across Cell Membranes Down Its Concentration Gradient – a Process Called Osmosis
o Water molecules are small and uncharged so can diffuse directly across bilayer (slowly)
o Some cells contain specialized channel proteins called aquaporins in membrane, which facilitate the flow
o The total conc. of solute particles inside the call – also called osmolarity – generally exceeds solute conc. outside the cell
o Osmotic gradient pulls water into cell, down its conc. gradient – from area of low solute conc. (high water conc.) to an area of
high solute conc. (low water conc.) = osmosis
o If osmosis occurs without constraint, can swell the cell
Transporters and Their Functions
- Transporters are responsible for movement of most small, water-soluble, organic molecules and some inorganic ions across
membrane
- Each transporter is highly selective (often caters to only one type of molecule)
- Passive Transporters Move a Solute Along Its Electrochemical Gradient
o A transporter that mediates passive transport example is the glucose transporter
o This transporter consists of a polypeptide chain that crosses membrane at least 12 times and can adopt conformations and
switches reversibly and randomly between them
o One conformation exposes binding sites for glucose to exterior of cell; in another, it exposes the sites to cell interior
o Since glucose uncharged, its movement depends solely on conc. gradient
o When blood glucose levels are low (when hungry) – hormone glucagon stimulates liver cells to produce large amounts of
glucose by breakdown of glycogen
o Highly selective – binding sites in glucose transporter bind only to D-glucose and not its mirror image, L-glucose, which cell
cannot use for glycolysis
- Pumps Actively Transport a Solute Against Its Electrochemical Gradient
o For active transport, cells depend on transmembrane pumps, which can carry out active transport in 3 ways
1. ATP-driven pumps hydrolyze ATP to drive uphill transport
2. Coupled pumps link uphill transport of one solute across a membrane to downhill transport of another
3. Light-driven pumps, mainly in bacterial cells, use energy from sunlight to drive uphill transport
- The Na+ Pump in Animal Cells Uses Energy Supplied by ATP to Expel Na+ and Bring in K+
o Na+ pump uses energy derived from ATP hydrolysis to transport Na+ out of the cell as it carries K+ in (aka Na+-K+ ATPase or
Na+-K+ pump)
o Energy from ATP hydrolysis induces series of protein conformational changes that drive Na/K ion exchange
o The phosphate removed from ATP gets transferred to pump itself
o Na+ out and K+ in involves reaction cycle, in which each step depends on one before
o Tight coupling between steps in the pumping cycle ensures that pump operates only when appropriate ions available to be
transported, avoiding useless ATP hydrolysis
- The Na+ Pump Generates a Steep Concentration Gradient of Na+ Across Plasma Membrane
o Na+ pump ceaselessly expels Na+ that’s constantly entering the cell through other transporters and ion channels in membrane
o This way, pump keeps Na+ conc. in cytosol 10-30 times lower than extracellular fluid and K+ conc. 10-30 times higher
o Steep conc. gradient of Na+ across membrane acts with membrane potential to create large Na+ electrochemical gradient,
which tends to pull Na+ back into the cell
- Ca2+ Pumps Keep the Cytosolic Ca2+ Concentration Low
o Ca2+ kept at a low conc. in the cytosol compared to extracellular fluid
o An influx of Ca2+ into cytosol through Ca2+ channels is used by different cells as an intracellular signal to trigger cell processes
(like muscle contraction and fertilization)
o Eukaryotic cells maintain a low conc. of free Ca2+ in cytosol that is achieved mainly by means of ATP-driven Ca2+ pumps in
both plasma membrane and ER membrane, which actively pumps Ca2+ out of the cytosol
o Ca2+ pumps are ATPases that act similarly to Na+ pump. Main difference is that Ca2+ pumps return to original conformation
without requirement for binding and transporting a second ion
- Coupled Pumps Exploit Solute Gradients to Mediate Active Transport
o Electrochemical gradient of any solute across a membrane can be used to drive active transport of a second molecule
o Downhill movement of first solute down its gradient provides energy to power the uphill transport of the second = coupled
pumps
o If pump moves solutes in same direction it is a symport; if pump moves them in opposite directions it is a antiport
o A transporter that ferries only one type of solute (not coupled) is a uniport
- The Electrochemical Na+ Gradient Drives Coupled Pumps in the Membrane of Animal Cells
o Symports that make use of inward flow of Na+ down its steep electrochemical gradient have important role of driving import
of other solutes into animal cells
o Glucose-Na+ symport takes up glucose from gut lumen, even when conc. of glucose is higher in cell’s cytosol than in gut
lumen. Because electrochemical gradient for Na+ is steep, when Na+ moves into the cell down its gradient, glucose is “dragged”
into cell too.
o Two types of glucose transporters located at opposite ends of cell
o In apical, which faces gut lumen, has glucose-Na+ symports that take up glucose actively, creating a high glucose conc. in
cytosol
o In basal and lateral, cells have passive glucose uniports, which release glucose down its conc. gradient for use by other tissues
o Na+ driven pumps that operate as antiports are also important: Na+-H+ exchanger use downhill influx of Na+ to pump H+ out
of cell to control pH in cytosol, preventing cell interior from becoming too acidic
- Electrochemical H+ Gradients Drive Coupled Pumps in Plants, Fungi, and BacteriaDo not have Na+ pumps so rely mainly on
electrochemical gradient of H+ to import solutes into cell
o Gradient created by H+ pumps that pump H+ out of cell, setting up an electrochemical proton gradient and creating an acid pH
in the medium surrounding the cell
o In some photosynthetic bacteria, H+ gradient created by activity of light-driven H+ pumps such as bacteriorhodopsin
o In other bacteria, fungi, and plants, H+ gradient generated by H+ pumps that use energy of ATP hydrolysis to pump H+ out of
cell
o Different ATP-dependent H+ pump in lysosomes of animal cells and central vacuole of
plant and fungal cells that actively transport H+ out of cytosol into organelle, helping keep the pH of cytosol neutral and pH of
interior acidic
Ion Channels and the Membrane Potential
- Channels form transmembrane pores that allow passive movement of small water-soluble molecules into or out of cell or
organelle
- Narrow and highly selective pores
- Ion Channels Are Ion-Selective and Gated
o Ion channels shows ion selectivity, permitting some inorganic ions to pass but not others.
Depends on diameter and shape of ion channel and distribution of charged amino acids that line it.
o Ion channels narrow enough in places to force ions into contact with the channel wall so that only those ions of appropriate
size and charge are able to pass.
o Ion channels are not continuously open (most are gated: a specific stimulus triggers them to switch between a closed and an
open state by a change in their conformation)
o Unlike transporter, ion channel doesn’t need to undergo conformational changes with each ion it passes, having an advantage
over a transporter with respect to rate of transport
- Membrane Potential is Governed by the Permeability of a Membrane to Specific Ions
o Electrical changes mediated by alterations in permeability of membranes to ions
o Membrane also contains K+ leak channels that randomly change between open and closed states no matter the conditions
inside and outside of cell. When open, allows K+ to move freely. In resting cell, these are main ion channels open in membrane,
rendering membrane much more permeable to K+ than other ions
o When open, allows K+ to flow out of cell, leaving behind unbalanced negative charged on other side, creating a voltage
difference or membrane potential
o This charge imbalance prevents further K+ movement out of cell, establishing an equilibrium condition in which the membrane
potential keeping K+ inside is just strong enough to counteract tendency of K+ to move down its conc. gradient and out of cell
o In this equilibrium, electrochemical gradient for K+ is zero, even though much higher conc. of K+ inside the cell than out
o The membrane potential in steady-state conditions – in which flow of positive and negative ions across membrane is balanced,
so that no further difference in charge accumulates across membrane = resting membrane potential
o Nernst equation (V=62log(Co/Ci) expresses equilibrium quantitatively and can calculate theoretical resting membrane
potential if the ion conc. on either side of membrane are known
o Membrane potential at any time depends on both state of membrane’s ion channels and the ion concentrations on either side
of the membrane
- Ion Channels Randomly Snap Between Open and Closed States
o In patch-clamp recording, a fine glass tube is used as a microelectrode to isolate and make electrical contact with a small area
of the membrane at the surface of cell. Makes it possible to record activity of ion channels
By varying conc. of ions on either side of the patch, can test which ions go through the channels in the patch
o The voltage across the membrane patch (membrane potential) can be set and “clamped” at any chosen value
o Ability to expose membrane to different voltages allows us to see how changes in membrane potential affect the opening and
closing of ion channels in membrane
o Revealed that channels has moving parts and is snapping back and forth between open and closed conformations as channel is
knocked from one conformation to other by random thermal movements of the molecules in the environment
o When appropriate conditions change, random behavior continues but remains either open or closed for longer periods of time
- Different Types of Stimuli Influence the Opening and Closing of Ion Channels
o Ion channels differ from one another primarily with respect to their ion selectivity – the type of ions allowed to pass – and
their gating – the conditions that influence their opening and closing
o A voltage-gated channel’s probability of being open controlled by membrane potential
o A ligand-gated channel’s opening controlled by binding of some molecule (ligand) to channel
o A mechanically-gated channel’s opening controlled by mechanical force applied to channel (ex: auditory hair cells in ear) Ion
Channels and Nerve Cell Signaling
- Action Potentials Are Mediated by Voltage-gated Cation Channels
o When neuron stimulated, membrane potential of membrane shifts to less negative value = depolarization
o If depolarized enough voltage-gated Na+ channels in membrane to open transiently at site, allowing small amount of Na+ to
enter cell down steep electrochemical gradient
o Influx of positive charge depolarizes membrane further, opening additional voltage-gated Na+ channels further
depolarization
o +40 mV is close to membrane potential at which electrochemical driving force for movement of Na+ across membrane is zero
– that is, at which the effects of membrane potential and conc. gradient for Na+ are equal and opposite, so that Na+ has no
further tendency to enter or leave cell
o Na+ channels have automatic inactivating mechanism, causing them to rapidly adopt a special inactivated conformation
(closed), even though membrane is still depolarized
o Na+ channels remain in inactivated state until membrane potential returned to initial negative value
o During an action potential, depolarized axonal membrane helped to return to resting potential by opening of voltage-gated K+
channels
o As local depolarization reaches its peak, K+ ions start to flow out of cell through newly opened K+ channels down their
electrochemical gradient
o This rapid outflow of K+ through voltage-gated K+ channels brings membrane back to resting state
o Self-amplifying depolarization spreads outward: Na+ flowing in through open Na+ channels begins to depolarize neighboring
region of the membrane, allowing action potential to spread outward, eventually reaching axon terminals
- Voltage-gated Ca2+ Channels in Nerve Terminals Convert an Electrical Signal into a Chemical Signal
o Signal transmitted to target cells at specialized junctions = synapses
o Presynaptic and postsynaptic cells separated by synaptic cleft, which electrical signal cannot cross
Electrical signal converted to chemical in form of small, secreted signal molecules known as neurotransmitters that are stored in
nerve terminals within membrane-enclosed synaptic vesicles
o When action potential reaches nerve terminal, synaptic vesicles fuse with membrane, releasing neurotransmitters into
synaptic cleft
o Depolarization of nerve terminal membrane caused by arrival of action potential transiently opens voltage-gated Ca2+
channels concentrated in membrane of presynaptic nerve terminal. Ca2+ rushes into nerve terminal through open channels,
increasing its conc. in cytosol of the terminal. This immediately triggers membrane fusion that releases neurotransmitters
- Transmitter-gated Ion Channels in Postsynaptic Membrane Convert Chemical Signal Back into an Electrical Signal
o Released neurotransmitter binds to neurotransmitter receptors in postsynaptic membrane of target cell, changing membrane
potential of target cell, which, if large enough, triggers cell to fire an action potential
o Neurotransmitter then removed from synaptic cleft – either by enzymes that destroy it, by pumping it back into nerve
terminals that released it, or by uptake into neighboring non-neuronal cells
o Rapid responses depend on receptors that are transmitter-gated ion channels that converts chemical signal carried by NT back
into an electrical one. Channels open transiently in response to binding of NT, changing ion permeability of postsynaptic
membrane change in membrane potential
o If change big enough it will depolarize postsynaptic membrane and trigger an action potential in postsynaptic cell
- Neurotransmitter Can Be Excitatory or Inhibitory
o The receptor recognizes the NT that determines how the postsynaptic cell responds
o Excitatory NT receptors (acetylcholine and glutamate), are ligand-gated channels. When a NT binds, these channels open to
allow influx of Na+, depolarizing membrane and activating postsynaptic cell action potential
o Inhibitory NT receptors (GABA and glycine) are ligand-gated Cl- channels. When NT binds, these channels open, increasing
membrane permeability to Cl-, inhibiting postsynaptic cell by making its membrane harder to depolarize
- Most Psychoactive Drugs Affect Synaptic Signaling by Binding to Neurotransmitter Receptors
o Antidepressants block Na+ driven symport responsible for reuptake of excitatory NT serotonin, increasing amount of serotonin
available in the synapses that use it
o Sedatives bind to GABA-gated Cl- channels making the channels easier to open by GABA, rendering the neuron more sensitive
to GABA’s inhibitory action
- The Complexity of Synaptic Signaling Enables Us to Think, Act, Learn, and Remember
o A synapse can adjust magnitude of its response – reacting more vigorously (or less) to an incoming action potential – based on
how heavily the synapse has been used in the past = synaptic plasticity: triggered by entry of Ca2+ through special cation
channels in postsynaptic membrane, which can lead to functional alterations on either side of synapse
– in the amount of NT released from axon terminal, the way the postsynaptic cell responds to NT, or both
- Optogenetics Uses Light-gated Ion Channels to Transiently Activate or Inactivate Neurons in Living Animals
o Photosynthetic algae use light-gated channels to sense and navigate towards sunlight
Chapter 13 – How Cells Obtain Energy from Food
Lecture 14
- Cellular respiration is when an organism’s cells harvest energy from chemical-bond energy in sugars as the sugar molecule is
broken down and oxidized to CO2 and H2O
- Energy released during these reactions is captured in high-energy chemical bonds – covalent bonds that release a lot of energy
when hydrolyzed – in activated carriers (ATP/NADH)
- Carriers serve as portable sources of chemical groups and electrons needed for biosynthesis
The Breakdown and Utilization of Sugars and Fats
- Due to enzymes, cells degrade each glucose molecule step by step, paying out energy in small packets to activated carriers by
means of coupled reactions
- Energetically favorable, enzyme-catalyzed reactions involved in breakdown of foods are directly coupled to energetically
unfavorable reaction of ADP + P ATP
- Most ATP synthesis requires an intermediary.
- In 2nd pathway to making ATP, energy from other activated carriers used to drive ATP production = oxidative phosphorylation
in inner mitochondrial membrane
- Food Molecules are Broken Down in 3 Stages
o The breakdown process in which enzymes degrade complex organic molecules into simpler ones is called catabolism
o Stage 1: Enzymes convert large polymeric molecules in food into simpler monomeric subunits: proteins amino acids,
polysaccharides sugar, and fats fatty acids/glycerol. Also called digestion, occurs outside cells (intestine) or in specialized
organelles (lysosomes). After, small organic molecules enter cytosol of a cell, where gradual oxidative breakdown begins.
o Stage 2: Chain of reactions called glycolysis splits each glucose into two smaller
molecules of pyruvate. Takes place in the cytosol and generates 2 activated carriers: ATP and NADH. Pyruvate is transported
from cytosol into mitochondrial matrix, where giant enzyme complex converts each pyruvate into CO2 and acetyl CoA, another
activated carrier, also produced by stepwise oxidative breakdown of fatty acids from fats.
o Stage 3: Entirely in mitochondria. Acetyl group in acetyl CoA transferred to an oxaloacetate molecule to form citrate, which
enters citric acid cycle where the transferred acetyl group is oxidized to CO2 and producing a lot of NADH. The high-energy
electrons from NADH are passed along a series of enzymes called an electron transport chain, where the energy released by
their transfer drives oxidative phosphorylation – a process that produces ATP and consumes O2. This is where majority of energy
is released.
- Glycolysis Extracts Energy from the Splitting of Sugar
o Central process of stage 2 of catabolism is the oxidative breakdown of glucose in the sequence of reactions called glycolysis.
o Glycolysis produces ATP without O2 and occurs in cytosol
o Glycolysis splits molecule of glucose to form 2 molecules pyruvate
o The chemical rearrangements that generate pyruvate releases energy because electrons in pyruvate are at a lower energy
state than those in glucose
o For each glucose in glycolysis, 2 ATP are consumed to provide energy needed
o In later steps of glycolysis, 4 molecules of ATP produced. Energy also captured in this “payoff phase” in form of 2 NADH
o Net gain: 2 ATP and 2 NADH
- Glycolysis Produces Both ATP and NADH
o Pathway consists of 10 separate reactions, each producing a different sugar intermediate and each catalyzed by a different
enzyme
o Most energy released by breakdown of glucose is used to drive synthesis of ATP molecules from ADP and P = substrate level
phosphorylation because it occurs by the transfer of a phosphate group directly from a substrate molecule (sugar intermediate)
to ADP.
o Remainder of energy released during glycolysis is stored in electrons in NADH produced
by an oxidation: a hydrogen atom and an electron is removed from the sugar intermediate, glyceraldehyde 3-phosphate, and
transferred to NAD+, making NADH.
o NADH’s electron transfer in stage 3 to electron-transport chain in inner mitochondrial membrane occurs such that the
transfers release energy as the electrons fall from a state of higher energy to lower. (ultimately passed on to O2 H2O)
- Fermentations Can Produce ATP in Absence of Oxygen
o For many anaerobic organisms, glycolysis is principal source of ATP
o The pyruvate and NADH from glycolysis remains in cytosol
o Pyruvate is converted into products that are excreted from the cell: lactate in muscle cells or ethanol and CO2 in yeast cells
o NADH gives up its electrons in the cytosol and is converted back to NAD+ required to maintain the reactions of glycolysis
o Many bacteria and archaea can also generate ATP in absence of oxygen by anaerobic respiration, using a molecule other than
oxygen as a final electron acceptor
- Glycolytic Enzymes Couple Oxidation to Energy Storage in Activated Carriers
o The oxidation of glyceraldehyde 3-phosphate is coupled to form ATP and NADH in steps
6 and 7 of glycolysis
o Oxidizing the aldehyde group to a carboxylic acid group occurs in 2 steps
o Overall reaction releases enough free energy to transfer 2 electrons from the aldehyde to NAD+ NADH and to transfer a
phosphate group to a molecule of ADP ATP. It also releases enough heat to make overall reaction energetically favorable.
o The reaction is step 6 is the only one in glycolysis that creates a high energy phosphate linkage directly from inorganic
phosphate. This high energy linkage is generated in step 6 and consumed in step 7 to produce ATP.
- Several Organic Molecules Are Converted to Acetyl CoA in the Mitochondrial Matrix
o The pyruvate produced by glycolysis is actively pumped into mitochondrial matrix where it is rapidly decarboxylated by a
complex of 3 enzymes called the pyruvate dehydrogenase complex
o The products of pyruvate decarboxylation are CO2, NADH, and acetyl CoA
o Like the pyruvate derived from glycolysis, the fatty acids derived from fat are also converted into acetyl CoA in mitochondrial
matrix
o Fatty acids first activated by covalent linkage to CoA and are broken down completely by a cycle of reactions that trims 2
carbons at a time from their carboxyl end, generating one molecule of acetyl CoA for each turn of the cycle. NADH and FADH2
are produced.
o Amino acids can also be converted to acetyl CoA in mitochondrial matrix or another intermediate of citric acid cycle
- The Citric Acid Cycle Generates NADH by Oxidizing Acetyl Groups to CO2
o The citric acid cycle (Krebs cycle) accounts for 2/3 of total oxidation of carbon compounds with end products being CO2 and
high-energy electrons in form of NADH
o High-energy electrons from NADH are passed to electron transport chain in inner mitochondrial membrane
o At end of chain electrons combine with O2 to make H2O
o Doesn’t use O2 but is required as the final acceptor to allow NADH to get rid of its electrons and regenerate NAD+ to continue
cycle
o The citric acid cycle catalyzes the complete oxidation of carbon atoms of the acetyl groups in acetyl CoA, converting them to
CO2
o Acetyl group is transferred to a larger four-carbon molecule, oxaloacetate, to form six carbon tricarboxylic acid, citric acid
o Citric acid molecule (citrate) is oxidized, and the energy of this oxidation is harnessed to produce activated carriers
o Oxaloacetate that began process is regenerated at the end
o Each turn of the cycle also produces, in addition to 3 NADH, 1 FADH2 from FAD and 1
GTP from GDP
o The transfer of GTP’s terminal phosphate group to ADP ATP
o The energy stored in the readily transferred high-energy electrons of NADH and FADH2 is used to produce ATP through
oxidative phosphorylation on inner mitochondrial membrane, the only step that directly requires O2 in the atmosphere.
o The oxygen atoms required to make CO2 from the acetyl groups entering the citric acid cycle are supplied not by O2 but by
water
o Three molecules of water are split in each cycle, and the oxygen atoms of some of them are used to make CO2
o The O2 that we breathe is actually reduced to water by electron transport chain; it is not incorporated directly into the CO2 we
exhale
- Many Biosynthetic Pathways Begin with Glycolysis or the Citric Acid Cycle
o Many intermediates formed in glycolysis and citric acid cycle are siphoned off by anabolic pathways, in which they are
converted by series of enzyme-catalyzed reactions into amino acids, nucleotides, lipids, and other small organic molecules that
cell needs
- Electron Transport Drives the Synthesis of the Majority of the ATP in Most Cells
o The final stage in the oxidation of food molecules: oxidative phosphorylation
o Here is when the chemical energy captured by activated carriers produced during glycolysis and citric acid cycle is used to
generate ATP
o NADH and FADH2 transfers their high-energy electrons to the electron transport chain- a series of electron carriers embedded
in the inner mitochondrial membrane
o As electrons pass through series of electron acceptor and donor molecules that form the chain, they fall into successively
lower energy states
o The energy released is used to drive H+ across the inner membrane from mitochondrial matrix to the intermembrane space.
This movement generates a proton gradient across inner membrane, which serves as a source of energy that can be tapped to
drive energy-requiring reactions (phosphorylation of ADP to ATP)
o At end of chain, electrons added to O2 that have diffused into mitochondrion, and resulting reduced oxygen immediately
combines with H+ from surrounding H2O
o Electrons have now reached lowest energy level
o In total, complete oxidation of glucose to H2O and CO2 can produce about 30 ATP
Regulation of Metabolism
- Depending on conditions, a cell must decide whether to route key metabolites into anabolic or catabolic pathways – whether
to use to build molecules or burn to provide immediate energy
- Serve for immediate and long-term needs
- Catabolic and Anabolic Reactions Are Organized and Regulated
o Same substrates involved in many different pathways
o To balance interrelated reactions and to allow organisms to adapt to changes in food availability or energy expenditure, an
elaborate network of control mechanisms regulates and coordinates the activity of enzymes that catalyze the metabolic
reactions in a cell
o Binding of substrates to enzyme can either enhance its activity or inhibit it
- Feedback Regulation Allows Cells to Switch from Glucose Breakdown to Glucose Synthesis
o One way to increase available glucose is to synthesize it from pyruvate: gluconeogenesis
o It builds glucose from pyruvate
o Uses specific enzymes to bypass steps in glycolysis that are essentially irreversible
o The activity of the enzyme phosphofructokinase is allosterically regulated by binding of a variety of metabolites, which provide
both positive and negative feedback regulation
Enzyme is activated by byproducts of ATP hydrolysis- including ADP, AMP, and inorganic phosphate- and is inhibited by ATP.
o The enzyme that catalyzes reverse reaction, fructose 1,6-bisphosphatase, is regulated by the same molecules but in opposite
direction. This enzyme is activated when phosphofructokinase is turned off, allowing gluconeogenesis to proceed.
o Gluconeogenesis consumes 4 ATP and 2 GTP
- Cells Store Food Molecules in Special Reservoirs to Prepare for Periods of Need
o During food scarcity, gluconeogenesis is suppressed if alternatives available
o Fasting cells can mobilize glucose that has been stored in the form of glycogen, a branched polymer of glucose
o The synthesis and degradation of glycogen occur by separate metabolic pathways, which can be rapidly and coordinately
regulated according to need
o When more ATP is needed, cells break down glycogen in a reaction that is catalyzed by glycogen phosphorylase. Produces
glucose 1-phosphate, which is then converted to glucose 6-phosphate that feeds into glycolytic pathway
o Enzymes in each pathway are allosterically regulated by glucose 6-phosphate, but in opposite directions: glycogen synthetase
in the synthetic pathway is activated by glucose 6-phosphate, whereas glycogen phosphorylase (breaks down glycogen) is
inhibited by glucose 6-phosphate, as well as by ATP.
o Prevents glycogen breakdown when ATP is plentiful and favors its synthesis with glucose 6-phosphate conc. is high
o Most fat stored as droplets of water-insoluble triacylglycerols in specialized fat cells called adipocytes. In response to
hormonal signals, fatty acids can be released from these depots into bloodstream for cells to use as needed.
o A normal overnight fast mobilization of fat: in morning, most of the acetyl CoA that enters the citric acid cycle is derived from
fatty acids rather than glucose
o Excess glucose used to make glycogen or fat
o Plants convert some of the sugars they make through photosynthesis during daylight into fats and into starch, a branched
polymer of glucose very similar to animal glycogen
Chapter 14: Energy Generation in Mitochondria and Chloroplasts
Lecture 15
- Cells Obtain Most of Their Energy by a Membrane-based Mechanism
o Generation of ATP by oxidative phosphorylation requires a membrane
o 2 linked stages: one sets up an electrochemical proton gradient, the other uses that gradient to generate ATP. Both are carried
out by special protein complexes in membrane
Stage 1: high-energy electrons from oxidation of food, sunlight, or other sources are transferred along a series of electron
carriers (electron transport chain) embedded in the membrane. These transfers release energy that is used to pump protons,
derived from H2O, across the membrane and thus generate an electrochemical proton gradient. This is a form of stored energy
that can be harnessed when ions are allowed to flow back across membrane down their electrochemical gradient.
Stage 2: protons flow back down their gradient through a protein complex called ATP synthase, which catalyzes the energyrequiring synthesis of ATP from ADP and inorganic phosphate. This enzyme permits the proton gradient to make ATP.
o Due to chemiosmotic coupling, cells can harness energy of electron transfers to do work
- Chemiosmotic Coupling is an Ancient Process, Preserved in Present-Day Cells
o Chloroplasts and mitochondria evolved from bacteria that were engulfed by ancestral cells more than a billion years ago.
Evidence is that both reproduce in a manner similar tothat of most prokaryotes. Also harbor bacteria-like biosynthetic
machinery for making RNA and proteins, and they retain their own genomes
Mitochondria and Oxidative Phosphorylation
- Mitochondria produce bulk of cell’s ATP
- Patients with inherited disorder called myoclonic epilepsy and ragged red fiber disease (MERRF) are deficient in multiple
proteins required for electron transport muscle weakness, heart problems, epilepsy, and dementia.
- Mitochondria Can Change Their Shape, Location, and Number to Suit a Cell’s Needs
o In some cells, mitochondria remain fixed in one location, where they supply ATP directly to a site of unusually high energy
consumption
o In other cells, mitochondria fuse to form elongated, dynamic tubular networks, which are diffusely distributed through
cytoplasm. These are dynamic, continuously breaking apart by fission and fusing again.
- A Mitochondrion Contains an Outer Membrane, an Inner Membrane, and Two Internal Compartments
o The outer and inner membranes create 2 mitochondrial compartments: a large internal space called the matrix and a narrower
intermembrane space
o The outer membrane contains many molecules of transport protein called porin, which, forms wide aqueous channels through
the lipid bilayer. This makes intermembrane space chemically equivalent to the cytosol with respect to small molecules and
inorganic ions it contains
o The inner membrane is impermeable to the passage of ions and most small molecules, except where a path is provided by
specific membrane transport proteins.
o Mitochondrial matrix contains only molecules that are selectively transported into the matrix across inner membrane, and so
its contents are highly specialized
o Inner is site of oxidative phosphorylation and contains proteins of electron transport chain, the proton pumps, and the ATP
synthase needed for ATP production. Also contains transport proteins that allow entry of selected small molecules (pyruvate and
fatty acids that will be oxidized) into the matrix
o Inner membrane forms series of infoldings (cristae) that project into matrix space and increase surface area of membrane
- The Citric Acid Cycle Generates the High-Energy Electrons Required for ATP Production
o Both pyruvate produced by glycolysis, which takes place in cytosol, and the fatty acids derived from breakdown of fats can
enter mitochondrial intermembrane space through the porins in the outer mitochondrial membrane
o They are then transported across inner mitochondrial membrane into the matrix, where they are converted into acetyl CoA
o Acetyl groups in acetyl CoA is then oxidized to CO2 via citric acid cycle and activated carriers that enter electron transport
chain in inner mitochondrial membrane
- The Movement of Electrons is Coupled to the Pumping od Protons
o NADH and FADH2 donate high-energy electrons to electron transport chain in inner mitochondrial membrane, becoming
oxidized to NAD+ and FAD
o Electrons are quickly passed along chain to O2 to form H2O
o The stepwise movement of electrons releases energy that can be used to pump protons across the inner membrane. Resulting
proton gradient is used to drive synthesis of ATP
o The chemiosmotic mechanism for ATP synthesis is called oxidative phosphorylation, involving the consumption of O2 and
addition of a phosphate group to ADP ATP
o In photosynthesis, high-energy electrons come from organic green pigment chlorophyll, which captures energy from sunlight
o Single-celled organisms (archaea and bacteria) use inorganic substances like H, Fe, and S
- Protons Are Pumped Across Inner Mitochondrial Membrane by Proteins in Electron Transport Chain
o Each chain contains over 40 proteins, grouped into 3 large respiratory enzyme complexes that each contain multiple individual
proteins, including transmembrane proteins that anchor the complex in the inner mitochondrial membrane
o The 3 in order used are: NADH dehydrogenase complex, cytochrome c reductase complex, and cytochrome c oxidase complex
o Each contains metal ions and chemical groups that facilitate passage of electrons
o Movement of electrons through these series of complexes is accompanied by pumping of protons from mitochondrial matrix
to intermembrane space
o The first complex, NADH dehydrogenase, accepts electrons from NADH (extracted from NADH in form of hydride ion (H-),
which is converted into a proton and 2 high-energy electrons). H- H+ and 2e- catalyzed by NADH dehydrogenase complex
o Electrons then passed along chain to each other complexes using mobile electron carriers to ferry electrons between
complexes
o This transfer is energetically favorable: electrons passed from electron carriers with weaker electron affinity to those with
stronger electron affinity, until they combine with O2 H2O
- Proton Pumping Produces a Steep Electrochemical Proton Gradient Across Inner Mitochondrial Membrane
o Pumping of protons generates a H+ gradient – or pH gradient – across inner membrane the pH in matrix 0.7 higher than in the
intermembrane space
o Proton pumping generates a voltage gradient – or membrane potential – across inner membrane; as H+ flows outward, the
matrix side of the membrane becomes negative and the side facing the intermembrane space becomes positive
o The force that drives the passive flow of an ion across a membrane is proportional to the ion’s electrochemical gradient that
depends on both the voltage across membrane and the ions conc. gradient
o The inner mitochondrial membrane’s pH gradient and membrane potential works to create steep electrochemical proton
gradient, making it energetically favorable for H+ to flow back into mitochondrial matrix
- ATP Synthase Uses the Energy Stored in the Electrochemical Proton Gradient to Produce ATP
o Electrochemical proton gradient across inner mitochondrial membrane used to drive synthesis of ATP from ADP and P
o ATP synthase, a large, multisubunit protein embedded in inner mitochondrial membrane (found in animal cells, plants/algae,
and bacteria)
o The part of the protein that catalyzes phosphorylation of ADP projects into mitochondrial matrix and is attached by a central
stalk to a transmembrane H+ carrier
o Passage of protons through this carrier carrier and stalk to spin rapidly. As it rotates, it rubs against proteins in stationary
head, altering their conformation and prompting them to make ATP
o Chemical deformation gets converted into chemical-bond energy of ATP
o ATP synthase can operate in reverse – using the energy of ATP hydrolysis to pump protons uphill, against electrochemical
gradient across the membrane
o Its function depends on magnitude of gradient across membrane in which enzyme is embedded
- Coupled Transport Across Inner Mitochondrial Membrane Is Also Driven by the Electrochemical Proton Gradient
o Carrier proteins that bind to imported and exported molecules can couple their transport to the energetically favorable flow of
H+ into the matrix
o Pyruvate and P are ach co-transported inward along with protons, as the protons move down their electrochemical gradient
into the matrix
o Other transporters take advantage of membrane potential generated by electrochemical proton gradient, which makes the
matrix side of inner membrane more negative than side that faces intermembrane space
o Antiport carrier protein uses voltage gradient to export ATP from matrix and to bring ADP in
- The Rapid Conversion of ADP to ATP in Mitochondria Maintains a High ATP/ADP Ratio in Cells
o ADP molecules (made by hydrolysis of ATP in cytosol) are rapidly drawn back into mitochondria for recharging, while bulk of
ATP made in mitochondria are exported into cytosol
o Concentration of ATP in cytosol must be kept about 10 times higher than that of ADP
o Poison cyanide, blocks electron transport in inner mitochondrial membrane, causing cell death
- Cell Respiration Is Amazingly Efficient
o The oxidative pathways that allow cells to extract energy from food efficiently and in a usable form involve many
intermediates, each differing only slightly from predecessor
o Huge amounts of energy locked up in food molecules can be parceled out into small packets that can be captured in activated
carriers, such as NADH and FADH2
o As electrons pass from one enzyme complex to the next, they promote the pumping of protons across the inner mitochondrial
membrane at each step along the way
Each NADH molecule provides enough net energy 2.5 molecules ATP
o FADH2 bypass the NADH dehydrogenase complex and pass electrons to membrane-embedded mobile carrier ubiquinone
Since these electrons enter further down the chain than NADH, they promote pumping of fewer protons net 1.5 molecules ATP
Molecular Mechanisms of Electron Transport and Proton Pumping
- Protons Are Readily Moved by the Transfer of Electrons
o Hydrogen atoms are most abundant atom in living organisms
o Protons in water highly mobile – by rapidly dissociating from one water molecule and associating with its neighbor, they can
rapidly flit through a hydrogen-bonded network of water molecules
o Water serves as a ready reservoir for donating and accepting protons
o A membrane in which electrons are being passed along an electron-transport chain, it is a simple matter, to move protons
from one side of the membrane to another
o All that is needed is that the electrons carrier be oriented in the membrane in such a way that it accepts an electron – along
with a proton from water – on one side of the membrane, and then releases that proton on the other side of the membrane
when the electron is passed on to the next electron carrier molecule in the chain
- The Redox Potential Is a Measure of Electron Affinities
o The proteins in the respiratory chain guide the electrons so that they move sequentially from one enzyme complex to another
o Each electron transfer is an oxidation-reduction
o Molecule donating the electron becomes oxidized
o Receiving molecule becomes reduced
o Electrons pass spontaneously from molecules that have a low affinity for their outer-shell electrons, and thus lose them easily,
to molecules that have a higher affinity for electrons
o NADH has a low electron affinity, so that its electrons are readily passed to the NADH dehydrogenase complex
o Redox reactions proceed spontaneously depends on the free-energy change for the electron transfer, which depends on the
relative affinities of the two molecules for electrons
o Molecules that donate protons = acids
o Molecules that accept protons = bases
o These molecules exist in conjugate acid-base pairs
o NADH and NAD+ are redox pairs – NADH converted to NAD+ by loss of electrons
- Electron Transfers Release Large Amounts of Energy
o Amount of energy that can be released by an electron transfer can be determined by comparing the redox potentials of the
molecules involved
o 1:1 mixture of NADH and NAD+ has a redox potential of -320 mV, indicating that NADH has a weak affinity for electrons – and
a strong tendency to donate them
o 1:1 mixture H2O and ½ O2 has a redox potential of +820 mV, indicating that O2 has a strong affinity for electrons
o Difference in redox potential between these two pairs = 1140 mV, which means that the transfer of each electron from NADH
to O2 under these standard conditions is favorable
o If compared this free-energy change needed for the formation of phosphoanhydride bonds in ATP in cells, we see that enough
energy is released by the oxidation of one NADH molecule to synthesize a couple of molecules of ATP
o Transfer of electrons from NADH to O2 is made in many small steps along the ETC, enabling nearly half of the released energy
to be stored in the proton gradient across the inner membrane rather than getting lost to the environment as heat
- Metals Tightly Bound to Proteins Form Versatile Electron Carriers
o Each of 3 respiratory enzyme complexes includes metal atoms that are tightly bound to the proteins; once electron donated to
a respiratory complex, it moves within the complex by skipping from one embedded metal ion to another with a greater affinity
for electrons
o When passing from one complex to the next, the electrons are ferried by electron carriers that diffuse freely within the lipid
bilayer
o Ubiquinone picks up electrons from the NADH dehydrogenase complex and delivers them to the cytochrome c reductase
complex
o Quinone functions similarly in photosynthesis
o Ubiquinone also serves as the entry point for electrons donated by FADH2 that is generated during the citric acid cycle and
from fatty acid oxidation
o Redox potential of different metal complexes influence where they are used along ETC
o Iron-sulfur centers have low affinity for electrons
In NADH dehydrogenase complex – passes electrons to ubiquinone and later in pathway, iron atoms held in the heme groups
bound to cytochrome proteins are commonly used as electron carriers
o Heme groups give cytochromes (cytochrome c reductase and c oxidase complexes) color
o Like electron carriers, cytochrome proteins increase in redox potential the further down the mitochondrial ETC they’re located
- Cytochrome c Oxidase Catalyzes the Reduction of Molecular Oxygen
o Cytochrome c oxidase, the final electron carrier in respiratory chain, has highest redox potential
o It removes electrons from cytochrome c, oxidizing it, and pass them off to O2 to produce H2O (4 electrons donated by
cytochrome x + 4 protons from aqueous added to each O2) 4 e- + 4H+ + O2 2H2O
o In addition to protons that combine with O2, 4 other protons pumped across membrane during transfer of 4 electrons from
cytochrome c to O2, driving allosteric changes in the conformation of the protein that move protons out of mitochondrial matrix
o Special oxygen-binding site within protein complex (has a heme group + Cu atom) serves as final repository for all electrons
donated by NADH at start of ETC – where almost all oxygen we breathe is consumed
o Once O2 picks up electron superoxide radical O2- that is dangerously reactive and will take up 3 electrons wherever it can
find them. This can cause damage to nearby DNA, proteins, and lipid membranes
o Active site of cytochrome c oxidase holds on tightly to an oxygen molecule until it receives all 4 electrons needed H2O
o Poisons (cyanide) are toxic because they bind tightly to cytochrome c oxidase complexes, halting ETC and production of ATP
Chloroplasts and Photosynthesis
- Photosynthesis – series of light-driven reactions that creates organic molecules from CO2
- Use electrons from water and energy of sunlight to convert CO2 organic compounds
- Carried out in specialized intracellular organelle – the chloroplast, which contains light-capturing pigments such as green
pigment chlorophyll
- Leaves major site; only occurs during daylight hours, producing ATP and NADH that can be used at any time of day to convert
CO2 into sugar inside the chloroplast – carbon fixation
- Chloroplasts Resemble Mitochondria but Have an Extra Compartment – the Thylakoid
o Have highly permeable outer membrane and less permeable inner membrane where membrane transport proteins are
embedded
o The inner membrane surrounds a large space called the stoma, analogous to mitochondrial matrix and contains metabolic
enzymes
o Chloroplast inner membrane doesn’t contain photosynthetic machinery (different mito)
o Light-capturing systems, ETC, and ATP synthase that produce ATP during photosynthesis are all in the thylakoid membrane –
folded to form a set of flattened sacs called the thylakoids, which are arranged in stacks called grana
o Space in each thylakoid is connected to other thylakoids, creating a 3 internal compartment, the thylakoid space, separate
from stroma
- Photosynthesis Generates – Then Consumes – ATP and NADPH
o Light energy + CO2 + H2O sugars + O2 + heat energy
o Stage 1: Light Reactions
Similar to oxidative phosphorylation in mitochondrial inner membrane ETC in thylakoid membrane harnesses the energy of
electron transport to pump protons into thylakoid space proton gradient that drives the synthesis of ATP by ATP synthase
High-energy electrons donated to photosynthetic ETC come from chlorophyll that has absorbed energy from sunlight
High-energy electrons that go down ETC in chloroplasts are donated not to O2, but NADP+ NADPH
o Stage 2: Dark Reactions
The produced ATP and NADPH used to drive manufacture of sugars from CO2
Carbon-fixation reactions can occur in absence of sunlight
Begin in chloroplast stroma, where they generate a three-carbon sugar called glyceraldehyde 3-phosphate that is exported to
cytosol, where it is used to make sucrose and large number of other organic molecules in leaves of plant
- Chlorophyll Molecules Absorb the Energy of Sunlight
o Chlorophylls best absorb light in the blue and red wavelengths
o Since these pigments absorb green light poorly, plants look green – green light reflected back to us
o When light of appropriate wavelength hits a molecule of chlorophyll, it excites electrons in diffuse network, perturbing the
way the electrons are distributed. This high-energy state is unstable, and an excited chlorophyll molecule will try to get rid of
this excess energy so it can return to stability
o Chlorophyll molecules in chloroplast able to convert light energy into a form of useful energy because they are associated with
special set of proteins in thylakoid membrane
- Excited Chlorophyll Molecules Funnel Energy into a Reaction Center
o Chlorophyll molecules held in large multiprotein complexes called photosystems that each consist of a set of antenna
complexes, that capture light energy, and a reaction center which converts it into chemical energy
o Each antenna complex has hundreds of chlorophyll molecules arranged so that light energy captured by one can be
transferred to neighboring one in the network
o Wandering energy will encounter a chlorophyll dimer called the special pair, which holds its electrons at a lower energy than
chlorophyll molecules
o Special pair located in reaction center – a transmembrane complex of proteins and pigments – and is positioned directly next
to a set of electron carriers ready to accept a high-energy electron from excited chlorophyll special pair
o As high-energy electron handed off, chlorophyll special pair becomes positively charged, and the electron carrier that accepts
the electron becomes negatively charged
o Rapid movement of this electron along a set of electron carriers in reaction center creates a charge separation that sets in
motion the flow of electrons from reaction center to ETC
- A Pair of Photosystems Cooperate to Generate Both ATP and NADPH
o The first photosystem (II) absorbs light energy its reaction center passes electrons to a
mobile electron carrier called plastoquinone (part of ETC) that transfers high-energy
electrons to a proton pump that uses the movement of electrons to generate an electrochemical proton gradient that then
drives the production of ATP by an ATP synthase in the thylakoid membrane
o At same time, photosystem I has been capturing energy from sunlight. Its reaction center passes high-energy electrons to
different mobile electron carrier, which brings them to an enzyme that uses them to reduce NADP+ to NADPH
o Combined action makes both ATP (photosystem II) and NADPH (photosystem I)
- Oxygen is Generated by a Water-Splitting Complex Associated with Photosystem II
o When mobile electron carrier removes electron from reaction center, it leaves behind a positively charged chlorophyll special
pair (missing electron must be replaced)
o Photosystem II – missing electron replaced by special protein complex that removes electron from water = water-splitting
enzyme that has cluster of Mg atoms that holds onto 2 water molecules from which electrons are extracted one at a time
o 4 electrons removed from 2 water molecules O2 released
o All oxygen in Earth’s atmosphere produced by water-splitting enzyme of photosystem II
- Special Pair of Photosystem I Receives its Electrons from Photosystem II
o Photosystem I gets electrons from II – the chlorophyll special pair in photosystem I serves as final electron acceptor for ETC
that carries electrons from photosystem II
o Electrons removed from water by photosystem II are passed through a proton pump to a mobile electron carrier called
plastocyanin that carries electrons to photosystem I, to replace the electrons lost by its excited chlorophyll special pair
o When light again absorbed by this photosystem, this electron boosted to high-energy level needed to reduce NADP+ to NADPH
o Extra boost of energy from the light by both photosystems allows an electron to be
moved from water (holds its electrons tightly) to NADPH (holds electron loosely)
o Energy left over to enable ETC that links the two photosystems to pump H+ across the thylakoid membrane, so that ATP
synthase can harness the light-derived energy for ATP production
- Carbon Fixation Uses ATP and NADPH to Convert CO2 into Sugars
o To provide energy and reducing power for cell, ATP and NADPH are used within the chloroplast stroma to produce sugars,
which can be exported by specific carrier proteins in the chloroplast inner membrane
o Production of sugar from CO2 and water (dark reactions) is called carbon fixation
o CO2 from atmosphere attached to 5-carbon sugar derivative, ribulose 1,5-biphosphate, to yield 2 molecules of 3-carbon
compound 3-phosphoglycerate
o Catalyzed in chloroplast stroma by enzyme called ribulose biphosphate carboxylase or Rubisco
o Fixation of CO2 catalyzed by Rubisco is energetically favorable because a continuous supply of energy-rich ribulose 1,5biphosphate is fed into it
o As compound consumed – by addition of CO2 – it must be replenished (energy from ATP and NADPH from light reactions)
o Series of reactions where CO2 combines with ribulose 1,5-biphosphate to make a simple sugar (also regenerates the ribulose)
= carbon-fixation cycle or Calvin Cycle
o Every 3 CO2 that enter 1 glyceraldehyde 3-phosphate - 9 ATP – 6 NADPH
- Sugars Generated by Carbon Fixation Can be Stored as Starch or Consumed to Produce ATP
o During excess photosynthetic activity, much of glyceraldehyde 3-phosphate is retained in chloroplast stroma and converted to
starch
o Other converted into fat in the stroma
o Both serve as energy reserves
o At night, stored starch and fat can be broken down to sugars and fatty acids, which are exported to cytosol to help support the
metabolic needs of the plant
o Some enter glycolytic pathway where it is converted to pyruvate
o Pyruvate + fatty acids can enter cell mitochondria and fed into citric acid cycle ATP by oxidative phosphorylation
o Can be converted into metabolites, including the disaccharide sucrose – major form in which sugar is transported between
cells of a plant (exported from the leaves via vascular bundle to provide carbohydrate to rest of plant)
The Evolution of Energy-Generating Systems
- Oxidative Phosphorylation Evolved in Stages
o Anaerobic fermentation reactions would have dumped organic acids (lactic or formic) into the environment that could have
lowered its environmental pH, favoring the survival of cells that evolved transmembrane proteins that could pump H+ out of
cytosol, preventing cell from becoming too acidic.
o One of these pumps may have used the energy available from ATP hydrolysis to eject H+ from the cell; such a proton pump
could have been the ancestor of present-day ATP synthases
o This need to conserve resources might have led to the evolution of electron-transport proteins that allowed cells to use the
movement of electrons between molecules of different redox potentials as a source of energy for pumping H+ across the
membrane
o Some of these cells might have used the nonfermentable organic acids that neighboring cells had excreted as waste to provide
the electrons needed to feed ETC
o Eventually, some bacteria would have developed H+-pumping electron- transport systems that were so efficient that they
could harvest more redox energy than they needed to maintain their internal pH
- Photosynthetic Bacteria Made Even Fewer Demands on Their Environment
o The major evolutionary breakthrough in energy metabolism - the formation of photochemical reaction centers that could use
the energy of sunlight to produce molecules such as NADH
o Occurred in ancestors of green sulfur bacteria
o Next step involved the evolution of organisms capable of using water instead of H2S as the electron source for photosynthesis.
This entailed the evolution of a water-splitting enzyme and the addition of a second photosystem, in conjunction with first, to
bridge the gap in redox potential between H2O and NADPH
o The availability of O2 made possible the development of bacteria that relied on aerobic metabolism to make their ATP.
o As organic materials accumulated as a by-product of photosynthesis, some photosynthetic bacteria lost their ability to survive
on light energy alone and come to rely on cell respiration
- The Lifestyle of Methanococcus Suggests That Chemiosmotic Coupling Is an Ancient Process
o Modern organisms that appear to be closely related to hypothetical cells from which all life evolved to live at 75 – 95 degrees
C, temperatures approaching boiling water
o Suggests common ancestor lived under very hot, anaerobic conditions
o Archaea that live in this environment today is Methanococcus jannaschii – isolated from hydrothermal vent a mile beneath
ocean surface, grows in complete absence of light and gaseous oxygen, using as nutrients the inorganic gases (H2, CO2, and N2)
that bubble up from vent
o Relies on N2 gas as source of nitrogen for making organic molecules such as amino acids
o Reduces N2 to ammonia (NH3) by addition of hydrogen = nitrogen fixation
o Energy for nitrogen fixation derived from transfer of electrons from H2 to CO2, with release of large amounts of CH4 as a
waste product
o Occurs in membrane pumping of protons across it electrochemical gradient driving an ATP synthase in same membrane to
make ATP
o The fact that such chemiosmotic coupling exists in an organism like Methanococcus suggests that the storage of energy in a
proton gradient derived from electron transport is
an extremely ancient process. Thus, chemiosmotic coupling may have fueled the evolution of nearly all life- forms on Earth.
Chapter 15 – Intracellular Compartments and Protein Transport
Lecture 16
- For a cell to operate effectively, the different intracellular processes that occur simultaneously must somehow be segregated
- Strategy by both prokaryotic and eukaryotic – aggregate the different enzymes required to catalyze a particular sequence of
reactions into large, multicomponent complexes
- Second strategy highly developed in eukaryotes – confine different metabolic processes, and proteins needed to perform
them, within different membrane-enclosed compartments
Membrane-Enclosed Organelles
- Eukaryotic cells are subdivided by internal membranes
- Eukaryotic Cells Contain a Basic Set of Membrane-Enclosed Organelles
o Membrane-enclosed organelles are surrounded by the cytosol, enclosed by PM
o Nucleus is surrounded by double membrane known as the nuclear envelope, and communicates with the cytosol via nuclear
pores that perforate the envelope
o Outer nuclear membrane is continuous with the membrane of the ER, a system of interconnected sacs and tubes of
membrane (site of synthesis of new membranes)
o Large areas of ER have ribosomes attached to cytosolic surface = rough ER
o Smooth ER lacks ribosomes – site of steroid hormone synthesis in some endocrine cells of the adrenal gland and site where
organic molecules are detoxified in liver cells (alc)
o The golgi apparatus receives proteins and lipids from ER, modifies them, and dispatches them to other destinations in the cell
o Lysosomes – intracellular degradation
o Endosomes – sorting od endocytosed material
o Mitochondria – ATP synthesis by oxidative phosphorylation
o Chloroplasts – ATP synthesis and carbon fixation by photosynthesis
o Peroxisomes – oxidation of toxic molecules
o Many membrane-enclosed organelles are held in relative locations in the cell by attachment to the cytoskeleton, especially to
microtubules
o Possible to separate one type of organelle from another by differential centrifugation
- Membrane-Enclosed Organelles Evolved in Different Ways
o Compartments probably evolved in stages
o Increase in size typical of eukaryotic cells probably could not have occurred without the development of internal membranes
o Nuclear membranes and the membranes of the ER, Golgi, endosomes, and lysosomes most likely originated by invagination of
the plasma membrane
o ER, Golgi, peroxisomes, endosomes, and lysosomes = endomembrane system
o Interiors of these organelles communicate with one another + with outside of the cell by means of small vesicles that bud off
from one of these organelles and fuse with another
o Mitochondria and chloroplast – possess own small genomes and make own proteins. The similarity of their genomes to
bacteria and resemblance of proteins to bacterial proteins suggest that they both evolved from bacteria engulfed by primitive
pre-eukaryotic cells with which they initially lived in symbiosis
Remain isolated from extensive vesicular traffic that connects interiors of most other membrane-enclosed organelles to one
another and to outside cell
Protein Sorting
- Before a eukaryotic cell divides, it must duplicate its membrane-enclosed organelles
- Newly synthesized proteins must be accurately delivered to their appropriate organelle
- Mitochondria, chloroplasts, peroxisomes, and interior of nucleus – proteins delivered directly from cytosol
- Golgi, lysosomes, endosomes, and inner nuclear membrane – proteins and lipids delivered indirectly via the ER (major site of
lipid and protein synthesis)
- Proteins Are Transported into Organelles by Three Mechanisms
o Synthesis of almost all proteins in the cell begins on ribosomes in cytosol; exceptions are the few mitochondrial and
chloroplast proteins synthesized on ribosomes inside them
o Fate of any protein synthesized in cytosol depends on amino acid sequence which contains a sorting signal that direct protein
to required organelle
o Ones that lack signal remain in cytosol
o When membrane-enclosed organelle imports a water-soluble protein to its interior it faces
a problem: transporting the protein across membrane, which are normally impermeable to hydrophilic macromolecules
From cytosol into nucleus – transported through selective gates called nuclear pores that penetrate inner and outer nuclear
membranes
From cytosol into ER, mitochondria, or chloroplast – transported across the organelle membrane by protein translocators in the
membrane. Transported protein usually unfolds in order to go through translocator. Moving onward from ER (or one
compartment of endomembrane system to another) – transported/ferried by transport vesicles that pinch off from the
membrane of one compartment and then fuse membrane of a 2nd compartment
- Signal Sequences Direct Proteins to the Correct Compartment
o The signal sequence is a continuous stretch of amino acid sequence and is often removed from the finished protein once it has
been sorted
o Necessary and sufficient to direct protein to destination
o Signal sequences specifying same destination can vary even though they have the same function: physical properties such as
hydrophobicity or the placement of charged amino acids often more important for the function of these signals than the exact
sequence
- Proteins Enter the Nucleus Through Nuclear Pores
o Nuclear envelope enclosed DNA and defines nuclear compartment
o Inner nuclear membrane – contains proteins that act as binding sites for chromosomes and others that provide anchorage for
nuclear lamina, a woven meshwork of protein filaments that lines inner face of membrane and provides structural support for
envelope
o Outer nuclear membrane – resembles membrane of ER, with which it is continuous
o Nuclear envelope in eukaryotic cells perforated by nuclear pores – gates for molecules
o Proteins that line the nuclear pore contain extensive, unstructured regions in which polypeptide chains are disordered that fill
the center of the channel, preventing passage of large molecules, but allowing small water-soluble ones to pass freely
o To gain entry to a pore, these large molecules display an appropriate sorting signal
(nuclear localization signal) that directs the protein and contains positively charged lysines or arginines
o Nuclear localization signal recognized by cytosolic proteins = nuclear import receptors
o They interact with fibrils that extend from rim of pore into cytosol
o It penetrates pore by grabbing onto repeated amino acid sequences within the tangle of nuclear pore proteins that fill the
center of the pore
o Nuclear import receptors interrupt interactions and open a local passageway through
meshwork and bump along from one repeat sequence to next, until they enter nucleus and deliver cargo
o Empty receptor returns to cytosol via nuclear pore for reuse
o Energy provided by hydrolysis of GTP, mediated by monomeric GTPase named Ran
o Nuclear pores transport proteins in their fully folded conformation and ribosomal components as assembled particles
o Proteins have to unfold to cross membranes of mitochondria and chloroplasts
- Proteins Unfold to Enter Mitochondria and Chloroplasts
o Chloroplasts have 3rd membrane system = thylakoid membrane
o Proteins imported have signal sequence at their N-terminus that allows them to enter
o Destined proteins translocated simultaneously across both inner and outer membranes at specialized sites where the 2
membranes contact each other
o Each unfolded as transported, and signal sequence removed after translocation
o Chaperone proteins inside organelles pull protein across 2 membranes and to fold it again
o Subsequent transport to particular site in organelle requires further sorting signals in the protein, which are exposed after the
signal sequence is removed
o Most membrane phospholipids are thought to be imported from ER and are transported by lipid-carrying proteins that extract
a phospholipid molecule from one membrane and deliver it to another
- Proteins Enter Peroxisomes from Both the Cytosol and the Endoplasmic Reticulum
o Peroxisomes contain one or more enzymes that produce hydrogen peroxide
o Break down variety of molecules – toxins, alcohol, fatty acids
o Synthesize phospholipids – including ones in myelin sheath
o Short sequence of 3 amino acids = import signal for peroxisomal proteins that is
recognized by receptor proteins in the cytosol that escorts cargo protein to peroxisome before returning to cytosol
o Peroxisomal membrane contains protein translocator that aids in their transport – but proteins don’t need to unfold to enter
peroxisome
o A few membrane proteins arrive via vesicles that bud from ER membrane and fuse with preexisting peroxisomes or import
proteins from cytosol to grow into mature peroxisome
- Proteins Enter the Endoplasmic Reticulum While Being Synthesized
o ER serves as an entry point for proteins for other organelles, as well as for ER itself
o Proteins destined for Golgi, endosomes, lysosomes, and cell surface all first enter ER
o One inside ER lumen or embedded in membrane, proteins will be ferried by transport vesicles from organelle to organelle
within endomembrane system, or to PM
o 2 kinds of proteins transferred from cytosol to ER: (1) water-soluble completely translocated across ER membrane and
released into ER lumen; (2) prospective transmembrane proteins partly translocated across ER membrane and become
embedded
o These 2 kinds initially directed to ER by an ER signal sequence
o Most proteins that enter ER begin to be threaded across ER membrane before polypeptide chain has been completely
synthesized – required ribosome attached to ER membrane
o Membrane-bound ribosomes attached to cytosolic side of ER membrane and make proteins that are being translocated to the
ER
o Free ribosomes are unattached to any membrane and make all of the other proteins encoded by the nuclear DNA
o Structurally and functionally identical
o Because proteins with ER signal sequence translocated as being made, no additional energy needed for transport; the
elongation of each polypeptide gives the thrust needed to push growing chain through ER membrane
o An mRNA molecule encoding a protein with an ER signal sequence, the polyribosome becomes riveted to ER membrane by
growing polypeptide chains, which have become inserted into the ER membrane
- Soluble Proteins Made on the ER are Released into the ER Lumen
o 2 protein components guide ER signal sequences to ER membrane: (1) signal-recognition particle (SRP) in cytosol binds to
ribosome and ER signal sequence when it emerges from ribosome and (2) SRP receptor embedded in ER membrane recognizes
SRP
o Once SRP engages with SRP receptor on the ER, the SRP is released and the receptor passes the ribosome to a protein
translocator in ER membrane, recommencing synthesis
o Polypeptide threaded across ER membrane through a channel in the translocator
o The signal sequence (N-terminus for soluble proteins, end synthesized first) opens the
channel in the protein translocator and remains bound to channel while rest of polypeptide chain is threaded though membrane
as a large loop
o Removed by transmembrane signal peptidase that has active site facing lumenal side
o Cleaved signal released from translocation channel into lipid bilayer and rapidly degrades
o Once C-terminus passed through translocation channel, it is released into ER lumen
- Start and Stop Signals Determine the Arrangement of a Transmembrane Protein in the Lipid Bilayer
o N-terminal signal sequence initiates translocation
o Transfer process halted by an additional sequence of hydrophobic amino acids, a stop-transfer sequence, further along the
polypeptide chain translocation channel to release growing polypeptide chain side-ways into lipid bilayer
o N-terminal sequence cleaved off and stop-transfer remains in bilayer form alpha helical membrane-spanning segment that
anchors protein in membrane protein ending up as single-pass transmembrane protein
o N-terminus lies on lumenal side of lipid bilayer and C-terminus on cytosolic side
o Sometimes internal signal sequence (start-transfer sequence) starts protein transfer and is never removed (unlike N-terminal
signal sequence) Usually occurs in which polypeptide chain passes back and forth across lipid bilayer
o Multipass membrane proteins are stitched into lipid bilayer as they are being synthesized
Vesicular Transport
Entry into ER lumen or membrane usually leads to entry in Golgi apparatus where proteins are modified and sorted for shipment
to other sites
- Vesicular transport or continual budding or fusion of transport vesicles extends outward from ER to PM and inward from PM to
lysosomes and provides routes of communication between interior of cell and its surroundings
- As proteins/lipids transported undergo chemical modifications like adding carb side chains
- Transport Vesicles Carry Soluble Proteins and Membrane Between Compartments
o Major outward secretory pathway starts with synthesis of proteins on ER membrane and their entry into the ER, and it leads
through Golgi to cell surface at the Golgi, a side branch leads off through endosomes to lysosomes
o A major inward endocytic pathway, responsible for ingestion and degradation of extracellular molecules, moves materials
from the PM, through endosomes, to lysosomes
o Recognition events depend on proteins displayed on the surface of transport vesicle
o Different types of transport vesicles shuttle between the various organelles, each carrying a distinct set of molecules
- Vesicle Budding Is Driven by the Assembly of a Protein Coat
o Vesicles that bud from membranes usually have a distinctive protein coat on cytosolic surface = coated vesicles
o After budding from parent organelle, vesicle sheds its coat, allowing its membrane to interact with membrane to which it will
fuse
o The coat helps shape membrane into a bud and captures molecule for onward transport
o Clathrin coated vesicles bud from both the Golgi on the outward secretory pathway and from the PM on the inward endocytic
pathway
o Clathrin molecules assemble into basketlike network on cytosolic surface of membrane and starts the shaping of the
membrane into a vesicle and a small GTP-binding protein, dynamin, assembles around each invaginated coated pit ring to
constrict pinching off the vesicle from its parent membrane
o A second class of coat proteins called adaptins secure the clathrin coat to the vesicle membrane and help select cargo
molecules for transport
o Molecules for onward transport carry specific transport signals that are recognized by cargo receptors in the Golgi or plasma
membrane. Adaptins help capture specific cargo molecules by trapping the cargo receptors that bind them.
- Vesicle Docking Depends on Tethers and SNAREs
o Often, vesicle actively transported by motor proteins that move along cytoskeletal fibers
o specificity of vesicular transport suggests that each type of transport vesicle displays molecular markers on its surface that
identify the vesicle according to its origin and cargo
o Recognized by complementary receptors on appropriate target membrane, including PM
o Identification process depends on diverse family of monomeric GTPases called Rab proteins. Specific Rab proteins on the
surface of each type of vesicle are recognized by corresponding tethering proteins on the cytosolic surface of the target
membrane.
o Additional recognition provided by a family of transmembrane proteins called SNAREs
o Once tethering protein captured a vesicle by grabbing hold of its Rab protein, SNAREs
on the vesicle interact with complementary SNAREs on the target membrane, docking vesicle in place
o SNAREs also play central role in catalyzing membrane fusion needed for a transport vesicle to deliver its cargo
o After docking, fusion of vesicle with its target membrane requires stimulatory signal
o Fusion requires for the two bilayers to come closer than just the protruding SNAREs to interact requiring water to be
displaced from hydrophilic surfaces of membrane (energetically unfavorable preventing random fusion)
o All membrane fusions must be catalyzed by SNARE proteins: the v-SNAREs and t-SNAREs wrap around each other acting like a
winch that pulls 2 bilayers closer
Secretory Pathways
- Newly made proteins, lipids, and carbohydrates are delivered from the ER, via the Golgi, to cell surface by transport vesicles
that fuse with PM in the process of exocytosis
- Each molecule that travels along this route passes through a fixed sequence of membrane-enclosed compartments and is
chemically modified en route
- Most Proteins Are Covalently Modified in the ER
o Disulfide bonds formed by the oxidation of pairs of cysteine side chains, a reaction catalyzed by an enzyme that resides in the
ER lumen; help stabilize structure of proteins that will encounter degradative enzymes and changes in pH outside the cell
o Many proteins that enter ER lumen or membrane are converted to glycoproteins in ER by covalent attachment of short
branched oligosaccharide side chains of multiple sugars = glycosylation by glycosylating enzymes in ER
o Oligosaccharides on proteins protect from degradation, hold it in the ER until folded, or help guide it to appropriate organelle
by serving as transport signal for packaging to appropriate vesicle (outer carbohydrate layer or glycocalyx for recognition)
o The oligosaccharide is originally attached to a specialized lipid, called dolichol, in the ER membrane; it is then transferred to
the amino (NH2) group of an asparagine side chain on
the protein, immediately after a target asparagine emerges in the ER lumen during protein translocation
o A simple sequence of three amino acids, of which the asparagine is one, defines which asparagines in a protein receive the
oligosaccharide.
o The addition of the oligosaccharide in the ER is only the first step in a series of further modifications before the mature
glycoprotein reaches the cell surface
o Oligosaccharide processing begins in the ER and continues in the Golgi apparatus
- Exit from the ER is Controlled to Ensure Protein Quality
o Proteins retained in ER by a C-terminal sequence of 4 amino acids = ER retention signal
o Chaperones hold proteins in ER until proper folding/assembly occurs
o If proper folding/assembly still fails, proteins exported to cytosol, where they’re degraded
- The Size of the ER is Controlled by the Demand for Protein
o If buildup of misfolded proteins is large enough, it triggers a complex program called unfolded protein response (UPR) that
prompts cell to make more ER, including chaperones and proteins concerned with quality control
o UPR allows a cell to adjust the size of its ER according to the load of proteins entering the secretory pathway
o In some cases, even an expanded ER cannot cope, and the UPR directs the cell to self-destruct through apoptosis
- Proteins Are Further Modified and Sorted in the Golgi Apparatus
o The golgi apparatus consists of flattened, membrane-enclosed sacs called cisternae
o Each Golgi stack has two distinct faces: an entry, or cis, face and an exit, or trans, face. The cis face is adjacent to the ER, while
the trans face points toward the PM
o Soluble proteins and membrane enter the cis Golgi network via transport vesicles derived from the ER
o Proteins travel through cisternae in sequence through transport vesicles that bud from one cisterna and fuse with next
o Proteins exit from trans Golgi network in transport vesicles destined for either the cell surface or another organelle of
endomembrane system
- Secretory Proteins are Released from the Cell by Exocytosis
o In all eukaryotic cells, a stream of vesicles buds from the trans Golgi network and fuses with the PM in the process of
exocytosis
o Supplies PM with newly made lipids and proteins (constitutive exocytosis pathway)
o Constitutive pathway also carries soluble to cell surface to be released to the outside in a process called secretion
o Entry into constitutive pathway doesn’t require signal sequence
o Also, a regulated exocytosis pathway that operates only in cells that are specified for secretion
o Each specialized secretory cell produces large quantities of a product (hormone, mucus) which is stored in secretory vesicles
for later release
o These vesicles bud off from trans Golgi and go near PM where they wait for the extracellular signal that will stimulate them to
fuse with PM and release their contents to the cell exterior by exocytosis
o Proteins destined for regulated secretion are sorted and packaged in trans Golgi network
o Proteins that travel by regulated pathway have special surface properties that cause them to aggregate with one another
under the ionic conditions (acidic pH/high Ca2+) in trans
o Proteins secreted by constitutive pathway don’t aggregate and are carried automatically to PM by transport vesicles
o Selective aggregation allows secretory proteins to be packaged into secretory vesicles at concentrations much higher than the
concentration of the unaggregated protein in Golgi lumen
Endocytic Pathways
- Eukaryotic cells are continually taking up fluid, as well as large and small molecules through endocytosis
- Material ingested is progressively enclosed by a small portion of PM, which first buds inward and then pinches off to form an
intracellular endocytic vesicle
- Ingested materials, including membrane components, are delivered to endosomes, from which they can be recycled to the PM
or sent to lysosomes for digestion
- Pinocytosis (cellular drinking) involves ingestion of fluid and molecules via small pinocytic vesicles
- Phagocytosis (cellular eating) involves ingestion of large particles, like microorganisms and cell debris, via large vesicles called
phagosomes
- Specialized Phagocytic Cells Ingest Large Particles
o Phagocytosis in protozoa is a form of feeding
o Phagosomes fuse with lysosomes, where food particles are digested
o Phagocytic cells – including macrophages – defend us against infection by ingesting invading microorganisms
o Particles must first bind to the phagocytic cell surface and activate one of many surface receptors (that can recognize
antibodies)
o Binding of antibody-coated bacteria to receptors induces phagocytic cell to extend sheetlike projections of PM, called
pseudopods, that engulf bacterium and fuse at their tips to form a phagosome
o Phagosome fuses with lysosome and microbe destroyed
- Fluid and Macromolecules are Taken Up by Pinocytosis
o Eukaryotic cells ingest bits of PM, along with small amounts of extracellular fluid, in the process of pinocytosis
o The rate at which the PM is internalized in pinocytic vesicles varies (large tho)
o Because a cell’s total surface area and volume remain unchanged during this process, as much membrane is being added to
the cell surface by exocytosis as is being removed by endocytosis
o Pinocytosis is carried out mainly by clathrin-coated pits and vesicles
o After they pinch off from PM, clathrin-coated vesicles rapidly shed coat and fuse with endosome
o Extracellular fluid is trapped in coated pit as it invaginates to form a coated vesicle, and so substances dissolved in the
extracellular fluid are internalized and delivered to endosomes. This fluid intake by clathrin-coated and other types of pinocytic
vesicles is balanced by fluid loss during exocytosis.
- Receptor-Mediated Endocytosis Provides a Specific Route into Animal Cells
o Pinocytosis is indiscriminate
o Clathrin-coated vesicles provide pathway for taking up specific macromolecules
o Macromolecules bind to complementary receptors on cell surface and enter cell as receptor-macromolecule complexes in
clathrin-coated vesicles = receptor-mediated endocytosis – provides selective concentrating mechanism that increases efficiency
of internalization of particular macromolecules
o Cholesterol is a lipid insoluble in water that is transported in bloodstream bound to protein in form of particles called LDL
o Cholesterol containing LDLs (secreted by liver) bind to receptors on cell surfaces receptor LDL complexes to be ingested by
receptor-mediated endocytosis and delivered to endosomes
o In acidic endosome, LDL dissociates from receptor (receptor returned in transport vesicle to PM for reuse, while LDL delivered
to lysosomes where broken down by hydrolytic enzymes)
o Cholesterol released into cytosol, where available for new membrane synthesis
- Endocytosed Macromolecules Are Sorted in Endosomes
o Most extracellular material taken up by pinocytosis is rapidly delivered to endosomes
o Endosomal compartment is complex set of connected membrane tubes and larger vesicles
o Early endosomes mature into late endosomes as they fuse with each other or with a preexisting late endosome
o Interior of endosome compartment is kept acidic (5-6) by ATP-driven H+ proton pump in the endosomal membrane that
pumps H+ into endosome lumen from cytosol
o The endosomal compartment acts as the main sorting station in the inward endocytic pathway, just as the trans Golgi network
serves this function in the outward secretory pathway
o Routes taken by receptors once entered an endosome differ according to type of receptor: (1) most returned to same PM
domain from which they came (LDL); (2) some travel to lysosomes, where they are degraded; (3) some proceed to a different
domain of PM, transferring bound cargo molecules across the cell from one space to another, a process called transcytosis
- Lysosomes Are the Principal Sites of Intracellular Digestion
o Many extracellular particles and molecules ingested by cells end up in lysosomes – a membranous sac of hydrolytic enzymes
that carry out controlled digestion of both extracellular materials and worn-out organelles
o Enzymes optimally active in acidic conditions (pH ~5) maintained in lysosomes
o Lysosomal membrane contains transporters that allow the final products of digestion of macromolecules to be transferred to
cytosol; from there, can be excreted or used by cell
o Membrane also contains ATP-driven H+ pump to maintain acidic pH and highly glycosylated to protect the proteins from
digestion by the lysosomal proteases
o The specialized digestive enzymes and membrane proteins of the lysosome are synthesized in the ER and transported through
the Golgi apparatus to the trans Golgi network
o Tagging permits the lysosomal enzymes to be sorted and packaged into transport vesicles, which bud off and deliver their
contents to lysosomes via endosomes
o Autophagy pathway is used to degrade obsolete parts of the cell by enclosure of organelle by a double membrane, creating a
autophagosome, which fuses with a lysosome
o Autophagy of organelles and cytosolic proteins increases when eukaryotic cells are starved or when they remodel themselves
extensively during development
Chapter 16 – Cell Signaling
Lecture 17
General Principles of Cell Signaling
- Process of conversion of signals is called signal transduction
- Signaling cell produces a particular type of extracellular signal molecule that is detected by the target cell
- Target cells possess protein called receptors that recognize and respond to signal molecule
- Signal transduction begins when receptor on target cell receives incoming extracellular signal and converts it to the
intracellular signaling molecules that alter cell behavior
- Signal reception and transduction = cell signaling
- Signals Can Act Over a Long or Short Range
o “public” style of cell-to-cell communication involves broadcasting the signal throughout the whole body by secreting it into the
bloodstream or plant’s sap
o Extracellular signal molecules used in this way are called hormones produced in endocrine cells of animals
o Less public process known as paracrine signaling where signal molecules diffuse locally
through the extracellular fluid, remaining in neighborhood to cell that secretes them, acting as local mediators on nearby cells
o Cells can respond to local mediators that they themselves produce, a form of paracrine signaling called autocrine signaling
o Neuronal signaling delivers messages over long distances – delivered quickly and specifically to individual target cells through
private lines
Electrical impulses converted into chemical form – each electrical impulse stimulates the nerve terminal to release a pulse of an
extracellular signal molecule called a neurotransmitter
o Most short-range of all doesn’t require the release of a secreted molecule but instead the cells make direct physical contact
through signal molecule in PM of signaling cell and receptor proteins embedded in PM of target cell (contact-dependent
signaling)
- Each Cell Responds to a Limited Set of Extracellular Signals, Depending on Its History and Current State
o Each receptor usually activated by only one type of signal
o Alters behavior of cell by changing its shape, movement, metabolism, or gene expression
o Signal from cell-surface receptor generally conveyed into target cell interior via a set of
intracellular signaling molecules that act in sequence and alter activity of effector proteins, those that have some direct effect
on behavior of target cell
o Extracellular signal molecule alone is not the message: the information conveyed by the signal depends on how the target cell
receives and interprets the signal
o Variety of receptors on cell makes the cell simultaneously sensitive to different extracellular signals and allows a small number
of signal molecules, used in different combinations, to exert subtle and complex control over cell behavior
o Presence of one signal modifies effects of another
o In absence of any signals, most animal cells are programmed to kill themselves
- A Cell’s Response to a Signal Can be Fast or Slow
o Rapid responses are possible because the signal affects the activity of proteins that are already present inside the target cell
o Slower responses due to the response to these extracellular signals that requires change in gene expression and production of
new proteins
- Some Hormones Cross the Plasma Membrane and Bind to Intracellular Receptors
o Extracellular signal molecules fall into 2 classes
o First and largest consists of molecules too large or hydrophilic to cross PM of target cell; they rely on receptors on surface of
target cell to relay message across membrane
o Second, and smaller, consists of molecules small or hydrophobic enough to pass through PM and into cytosol that will then
activate intracellular enzymes or bind to intracellular receptor proteins that regulate gene expression
o Steroid hormones are signal molecules that rely on intracellular receptor proteins
o Both cytosolic and nuclear receptors are nuclear receptors because when activated by hormone binding, they act as
transcription regulators in the nucleus – usually present in inactive form when not stimulated
o When hormone binds, receptor undergoes conformation change that activates the protein, allowing it to inhibit/promote
transcription of specific target genes
o Each hormone binds to different nuclear receptor, and each receptor acts at a different set of regulatory sites in DNA
o Receptor for testosterone required not just in one cell type to mediate one effect of
testosterone, but in many cell types to help produce the range of features that distinguish men from women
- Some Dissolved Gases Cross Plasma Membrane and Activate Intracellular Enzymes Directly
o Some dissolved gases can diffuse across membrane to cell interior and directly regulate activity of specific intracellular
proteins
o Nitric oxide (NO) is synthesized from amino acid arginine and diffuses readily from its site of synthesis into neighboring cells,
acting locally because it is quickly converted to nitrates and nitrites by reacting with oxygen and water outside cells
o Endothelial cells (line blood vessels) release NO in response to neurotransmitters secreted by nearby nerve endings, causing
smooth muscle cells in adjacent vessel wall to relax, allowing vessel to dilate so that blood flows through it more freely
o NO released by nerve terminals in penis acts as a local mediator to trigger blood-vessel dilation responsible for penile erection
o Inside many target cells, NO binds and activates enzyme guanylyl cyclase, stimulating formation of cyclic GMP from nucleotide
GTP
o Viagra enhances penile erection by blocking the enzyme that degrades cyclic GMP, prolonging NO signal
- Cell-Surface Receptors Relay Extracellular Signals via Intracellular Signaling Pathways
o Molecules too large or hydrophilic to cross PM of target cells bind to cell-surface receptor proteins that span PM
o Transmembrane receptors detect a signal on outside and relay message across the membrane into the interior of the cell
o Resulting intracellular signaling process works like relay race, in which message is passed downstream from one intracellular
signaling molecule to another, each activating or generating the next signaling molecule in the pathway, until metabolic enzyme
kicking into action: cytoskeleton into new conformation, or gene switched on/off
o Components of these intracellular signaling pathways perform many crucial functions:
Relay the signal onward and help spread it throughout the cell Amplify received signal Detect signals from more than one
intracellular signaling pathway and integrate them before relaying a signal onward
Distribute the signal to more than one effector protein branches in the information flow diagram and evoking a complex
response
o Steps in signaling pathway subject to modulation by feedback regulation
- Some Intracellular Signaling Proteins Act as Molecular Switches
o Many intracellular signaling proteins behave as molecular switches – receiving signal toggle from inactive form to an active
state
o For every inactivation step along the pathway, there has to be an inactivation mechanism
o Proteins that act as molecular switches fall into 2 classes
o First class (largest) consists of proteins that are activated/inactivated by phosphorylation.
Switch is thrown in one direction by a protein kinase, which covalently attaches a phosphate group onto the switch protein, and
in the other direction by a protein phosphatase, which takes the phosphate off again
Two main types of protein kinases operate in intracellular signaling: the most common are serine/threonine kinases, which
phosphorylates proteins on serines or threonines; others are tyrosine kinases, which does it on tyrosines
o Other class of switch proteins are GTP-binding proteins that go between active/inactive
depending on whether they have GTP or GDP bound to them Activated by GTP binding intrinsic GTP-hydrolyzing (GTPase)
activity Shut themselves off by hydrolyzing bound GTP to GDP
- Cell-Surface Receptors Fall into Three Main Classes
o Ion-channel-coupled receptors – change permeability of PM to selected ions, altering membrane potential, producing an
electrical current
o G-protein-coupled receptors – activate membrane-bound trimeric GTP-binding proteins which activate/inhibit an enzyme or
ion channel in PM, initiating intracellular signaling cascade
o Enzyme-coupled receptors – act as enzymes or associated with enzymes; when stimulated, enzymes activate a variety of
intracellular signaling pathways
o For many extracellular signal molecules there is more than one type of receptor, and these may belong to different receptor
classes
- Ion-Channel-Coupled Receptors Convert Chemical Signals into Electrical Ones
o Ion-channel-coupled receptors are responsible for rapid transmission of signals across
synapses in the nervous system
o Transduce a chemical signal, in form of neurotransmitter molecules, directly into an electrical signal, in the form of a change in
voltage across target cell’s PM
o Binding of NT alters conformation of receptor, so as to open an ion channel in PM, allowing flow of ions (Na+, K+, or Ca2+)
o Driven by electrochemical gradients, ions rush into or out of cell change in membrane potential that triggers a nerve impulse
or make it easier for other NT to do so
o G-protein-coupled receptors and enzyme-coupled receptors important for almost every cell type in the body
G-Protein-Coupled Receptors
- G-protein-coupled receptors (GCPR) (largest family of cell-surface receptors) mediate responses of extracellular signal
molecules, including hormones, local mediators, and NTs
- All GCPRs have a similar structure: each made of a single polypeptide chain that threads back and forth across the lipid bilayer
7 times
- Superfamily of seven-pass transmembrane receptor proteins
- Stimulation of GCPRs Activates G-Protein Subunits
o When an extracellular signal molecule binds to a GPCR, the receptor protein undergoes a conformational change that enables
it to activate a G protein located on the other side of the plasma membrane
o G-proteins composed of 3 protein subunits – alpha, beta, and gamma – two of which are tethered to the PM by short lipid tails
o In unstimulated state, the alpha subunit has GDP bound to it and the G protein is idle
o Receptor activates G protein by causing alpha subunit to decrease affinity for GDP, which is then exchanged for molecule of
GTP
o The two activated parts of G protein – the alpha subunit and beta-gamma complex – can then each interact directly with
target proteins in PM, which in turn may relay signal to other destinations in the cell
o Timing of activation is controlled by behavior of alpha subunit
o Alpha subunit has GTPase activity, and it eventually hydrolyzes its GTP to GDP, returning G protein to original, inactive
conformation
- Some G Proteins Directly Regulate Ion Channels
o Target proteins recognized by G-protein subunits are enzymes or ion channels in PM
o Binding of extracellular signal molecule to GPCR changes in activities of a specific subset of the possible target proteins in PM
response appropriate for signal of that type of cell
o G-protein regulation of ion channels in heartbeat of animals
- Many G-Proteins Activate Membrane-bound Enzymes that Make Small Messenger Molecules
o Two most frequent target enzymes for G proteins are adenylyl cyclase, which produces small intracellular signaling molecule
cyclic AMP, and phospholipase C, which generates the small intracellular signaling molecules inositol triphosphate and
diacylglycerol
o Adenylyl cyclase and phospholipase C activated by different types of G proteins, allowing cells to couple the production of
these small intracellular signaling molecules to different extracellular signals
o Small intracellular signaling molecules generated by enzymes are often called small messengers, or second messengers, that
when activated, generate a lot of small messengers that rapidly diffuse amplification and spreading of intracellular signal
- Cyclic AMP Signaling Pathway Can Activate Enzymes and Turn on Genes
o Many extracellular signals acting via GPCRs affect activity of enzyme adenylyl cyclase and thus alter intracellular concentration
of small messenger molecule cyclic AMP
o Activated G-protein alpha subunit switched on adenylyl cyclase increase in synthesis of cyclic AMP from ATP (G protein called
Gs)
o To terminate signal, enzyme called cyclic AMP phosphodiesterase, converts cyclic AMP to ordinary AMP (this enzyme
continuously active inside cell)
o Cyclic AMP exerts most of its effects by activating enzyme cyclic AMP-dependent protein kinase (PKA) that is normally inactive
in complex with regulatory protein
o Binding of cyclic AMP to regulatory protein conformational change that releases inhibition and unleashes active kinase
o Activated PKA catalyzes phosphorylation of particular serines or threonines on specific intracellular proteins, altering activity
of these target proteins
o In skeletal muscle, adrenaline increases cyclic AMP breakdown of glycogen (storage form of glucose) by activating PKA
activation of an enzyme that promotes glycogen breakdown and inhibition of an enzyme that drives glycogen synthesis
o In slow cyclic AMP responses, PKA phosphorylates transcription regulators that activate transcription of selected genes
- The Inositol Phospholipid Pathway Triggers a Rise in Intracellular Ca2+
o G-protein called Gq activates the membrane-bound enzyme phospholipase C propagation of signal by cleaving a lipid
molecule that is a component of PM
o The molecule is inositol phospholipid
o Signaling pathway begins with activation of phospholipase C which generates 2 small messenger molecules: inositol 1,4,5triphosphate (IP3) and diacylglycerol (DAG)
o IP3 is released into cytosol and binds to/opens Ca2+ channels embedded in ER membrane Ca2+ rushing into cytosol that then
signals other proteins
o DAG is embedded in PM and helps recruit and activate protein kinase C (PKC) – which needs to bind to Ca2+ to become active
– which translocates from the cytosol to the PM
o Once activated, PKC phosphorylates intracellular proteins that varies depending on cell
- A Ca2+ Signal Triggers Many Biological Processes
o Sperm fertilizes an egg Ca2+ channels to open to trigger egg development
o Ca2+ stimulates responses by binding to/influencing activity of Ca2+-responsive proteins
o Steep electrochemical gradient of Ca2+ exists across both the ER and PM
o Same pumps that normally keep cytosolic Ca2+ low help terminate Ca2+ signal
o When Ca2+ binds to calmodulin the protein undergoes conformational change enabling it to interact with target proteins and
altering their activities
o Class of target proteins for calmodulin is Ca2+/calmodulin-dependent protein kinases (CaM-kinases) when activated, influence
process in cell by phosphorylating proteins
- GPCR-Triggered Intracellular Signaling Cascades Can Achieve Astonishing Speed, Sensitivity, and Adaptability
o Fastest of all responses mediated by a GPCR = response of eye to light
o Rod photoreceptor cells in eye are responsible for noncolor vision in dim light
o In rod photoreceptor cell, light sensed by rhodopsin (G-protein-coupled light receptor)
o Light-activated rhodopsin activates G protein called transducing
o Activated alpha subunit of transducing activates intracellular signaling cascade cation channels to close PM of photoreceptor
cell change in voltage across cell membrane that alters NT release and leads to nerve impulse being sent to brain
o In bright sunlight, the signaling cascade undergoes a form of adaptation, stepping down amplification so that photoreceptor
cells aren’t overwhelmed and can still register increases and decreases in the strong light
o Adaptation occurs in intracellular signaling pathways that respond to extracellular signal molecules, allowing cells to respond
to fluctuations in concentration of molecules
o By taking advantage of + and – feedback mechanisms, adaptation allows a cell to respond both to messages that are
whispered and to those that are shouted
Enzyme-Coupled Receptors
- Like GPCR, enzyme-coupled receptors are transmembrane proteins that display their ligand-binding domains on outer surface
of PM
- Either acts on an enzyme itself or forms a complex with another protein that acts on an enzyme
- Role in responses to extracellular signal proteins (“growth factors”) that regulate growth, proliferation, differentiation, and
survival of cells in animal tissues
- Responses typically slow; lead to change in gene expression
- Largest class consists of receptors with a cytoplasmic domain that functions as a tyrosine protein kinase which phosphorylates
tyrosines on specific intracellular signaling proteins
- Receptors called receptor tyrosine kinases (RTKs)
- Activated RTKs Recruit a Complex of Intracellular Signaling Proteins
o Enzyme-coupled receptor proteins usually have only one transmembrane segment, which spans lipid bilayer as a single alpha
helix
o Binding of an extracellular signal molecule two receptor molecules to come together in PM dimer
o Brings two intracellular tails of receptors together, activating their kinase domains so that each tail phosphorylates the other
o Tyrosine phosphorylation triggers assembly of intracellular signaling complex on cytosolic tails of receptor
o Newly phosphorylated tyrosines serve as docking sites for intracellular signaling proteins
o Docked intracellular signaling proteins possess specialized interaction domain that recognizes specific phosphorylated
tyrosines on the receptor tails
o Signaling protein complexes assembled on cytosolic tails of RTKs can transmit a signal along several routes simultaneously to
many destinations in the cell, activating and coordinating numerous biochemical changes required to trigger a complex response
o To terminate process, tyrosine phosphatases remove phosphates that were added to the tyrosines of both of the RTKs and
other intracellular signaling proteins in response
- Most RTKs Activate the Monomeric GTPase Ras
o Ras – small GTP-binding protein bound by a lipid tail to the cytoplasmic face of PM
o Ras protein member of large family of small GTP-binding proteins called monomeric GTPases and resembles alpha subunit of a
G protein (functions as molecular switch)
o Ras cycles between 2 distinct conformational states – active when GTP is bound and inactive when GDP is bound
o Interacting with active protein called Ras-GEF Ras exchange GDP for GTP (active)
o Ras switched off by GAP protein called Ras-GAP that promotes hydrolysis of GTP
o When active, initiates phosphorylation cascade in which series of serine/threonine protein kinases phosphorylate and activate
one another in sequence
o Relay system carries signal from PM to nucleus and includes a three-protein-kinase molecule called MAP-kinase signaling
module and final kinase of chain is MAP kinase
o MAP kinase is phosphorylated/activated by enzyme called MAP kinase kinase, and this protein is switched on by MAP kinase
kinase kinase (which is activated by Ras)
o At end of MAP-kinase cascade, MAP kinase phosphorylated effector proteins, including transcription regulators, altering their
ability to control gene transcription
o May stimulate cell proliferation, cell survival, or cell differentiation
o Cancer
- RTKs Activate PI 3-Kinase to Produce Lipid Docking Sites in the Plasma Membrane
o Many extracellular signal proteins that stimulate animal cells to survive and grow act through RTKs
o Enzyme phosphoinositide 3-kinase (PI 3-kinase) phosphorylates inositol phospholipids in PM that serve as docking sites for
specific intracellular signaling proteins, which relocate from cytosol to PM, where they can activate one another
o Relocated signal protein Akt (protein kinase B (PKB)) promotes growth and survival of cells, inactivating the signaling proteins
it phosphorylates
o Akt phosphorylates and inactivates cytosolic protein Bad where when active promotes apoptosis
o PI-3-kinase-Akt signaling pathway also stimulates cells to grow in size by indirectly activating a large serine/threonine kinase
called Tor – which stimulates cells to grow by enhancing protein synthesis and inhibiting protein degradation
o Anticancer drug rapamycin inactivates Tor
- Some Receptors Activate a Fast Track to the Nucleus
o Direct route to control gene expression uses receptor for protein Notch
o Notch – the receptor acts as transcription regulator that activates by binding of Delta (transmembrane signal protein on
surface of neighboring cell)
o Notch receptor becomes cleaved release of cytosolic tail of receptor, now free to move to the nucleus where it helps activate
appropriate set of Notch-responsive genes
- Cell-Cell Communication Evolved Independently in Plants and Animals
o Like animals, plants make use of transmembrane cell-surface receptors (especially enzyme-coupled receptors)
o In contrast to animals, plant cells seem not to use RTKs, steroid-hormone-type nuclear receptors, or cyclic AMP, and seem to
use few GCPRs
o In plants for response of cells to ethylene, when empty the receptor is active, activating associated protein kinase that shuts
off ethylene-responsive genes in nucleus
o When ethylene present, receptor and kinase are inactive and ethylene-responsive genes are transcribed
o Common in plants: signals act to relieve transcriptional inhibition
- Protein Kinase Networks Integrate Information to Control Complex Cell Behaviors
o Most extensive links are those mediated by protein kinases present in each pathway
o Kinases often phosphorylate, and regulate, components in other signaling pathways, in addition to components in their own
pathway
o Crosstalk occurs between different pathways
o Integrating proteins can deliver signal to many downstream targets
Chapter 17 – Cytoskeleton
Lecture 18
- Ability of cells to adopt shapes, organize components in interior, interact mechanically with environment, and carry out
coordinated movements depends on cytoskeleton – an intricate network of protein filaments that extends throughout the
cytoplasm
- Cytoskeleton built on the framework of three types of protein filaments: intermediate filaments, microtubules, and actin
filaments
- Family of fibrous proteins forms intermediate filaments; globular tubulin subunits form microtubules; and globular actin
subunits form actin filaments
Intermediate Filaments
- Intermediate filaments main function is to enable cells to withstand the mechanical stress that occurs when cells are stretched
- Thinner than actin filaments and thicker than myosin filaments
- Toughest and most durable; found in cytoplasm of most animal cells
- Form network throughout cytoplasm, surrounding nucleus and extending to cell periphery
- Often anchored to PM at cell-cell junctions called desmosomes, where PM is connected to that of another cell
- Also found within nucleus forming a meshwork called nuclear lamina, which underlies and strengthens nuclear envelope
- Intermediate Filaments Are Strong and Ropelike
o Strands of cable made of intermediate filament proteins, fibrous subunits each containing central elongated rod domain with
distinct unstructured domains at either end
o Rod domain has extended alpha-helical region that enables pairs of intermediate filament proteins to form stable dimers by
wrapping around each other in coiled-coil configuration
o 2 dimers in opposite directions associate staggered tetramer
o Tetramers associate side-by-side and assemble ropelike intermediate filament
o All interactions between the intermediate filament proteins depend solely on noncovalent bonding; it is the combined
strength of the overlapping lateral interactions along the length of the proteins that gives intermediate filaments their strength
o Central rod domains of different intermediate filament proteins are similar in size and amino acid sequence, so they pack
together forming similar diameter and structure
o Terminal domains vary greatly in size and sequence – exposed to surface of filament where allowed to interact with specific
components in cytoplasm
- Intermediate Filaments Strengthen Cells Against Mechanical Stress
o Prominent in cytoplasm of cells subject to mechanical stress
o Four classes: (1) keratin filaments in epithelial cells; (2) vimentin and vimentin-related filaments in connective-tissue cells,
muscle cells, and supporting cells of nervous system (glial cells); (3) neurofilaments in nerve cells; (4) nuclear lamins, which
strengthen the nuclear envelope
o First three in cytoplasm, fourth in nucleus
o All formed by polymerization of their corresponding intermediate filament subunits
o Keratin filaments are most diverse class and are formed from mixture of different keratin subunits. They span interiors of
epithelial cells. The ends are anchored to desmosomes, and filaments associate laterally with other cell components through
globular head and tail domains that project from their surface. They distribute the stress that occurs when skin is stretched.
o Many intermediate filaments are further stabilized and reinforced by accessory proteins, such as plectin, that cross-link the
filaments into bundles and link them to microtubules, to actin filaments, and to adhesive structures in desmosomes,
- The Nuclear Envelope Is Supported by a Meshwork of Intermediate Filaments
o The intermediate filaments that form this tough nuclear lamina are constructed from a class of intermediate filament proteins
called lamins
o Nuclear lamina and cytoplasmic intermediate filaments disassemble in mitosis
o Disassembly and reassembly of nuclear lamina controlled by phosphorylation and dephosphorylation of the lamins
o Lamins phosphorylated by protein kinases conformational change that weakens the binding between the lamin tetramers
and causes filaments to fall apart
o Dephosphorylation by protein kinases at end of mitosis lamina to reassemble
o Defects in a particular nuclear lamin are associated with certain types of progeria—rare disorders that cause affected
individuals to age prematurely.
Microtubules
- Crucial organizing role in all eukaryotic cells
- Can rapidly disassemble in one location and reassemble in another
- Grow out from a small structure near center of the cell called the centrosome
- Create system of tracks within cell, along with vesicles, organelles, and others to be transported
- During mitosis, cytoplasmic microtubules disassemble and reassemble into an intricate structure called the mitotic spindle
- Can also form stable structures, such as cilia and flagella
- Microtubules are Hollow Tubes with Structurally Distinct Ends
o Microtubules are built from subunits – molecules of tubulins – each of which itself a dimer composed of two very similar
globular proteins called alpha and beta tubulin, bound tightly together by noncovalent interactions
o Tubulin dimers stack by noncovalent bonding to form wall of hollow cylindrical microtubule
o Each protofilament has a structural polarity with alpha tubulin (minus end) at one end and beta tubulin (plus end) at the other,
and this polarity – the directional arrow embodied in the structure – is the same for all the protofilaments, giving a structural
polarity to whole microtubule
o Tubulin dimers will add to either end of a growing microtubule, more rapidly to plus end
o If had no polarity, they could not guide intracellular transport
- The Centrosome Is the Major Microtubule-Organizing Center in Animal Cells
o In animal cells, microtubules grow from centrosome (close to nucleus when not in mitosis) and organizes an array of
microtubules that radiates outward through cytoplasm
o Centrosome consists of a pair of centrioles, surrounded by matrix of proteins that includes ring-shaped structures formed from
gamma tubulin, and each gamma tubulin ring complex serves as the starting point, or nucleation sire, for growth of microtubule
o Alpha beta tubulin dimers add to each gamma ring complex with minus end embedded in centrosome, and growth occurs only
at the plus end that extends to cytoplasm
o Each centriole sits perpendicular to its partner and is made of a cylindrical array of short microtubules
- Growing Microtubules Display Dynamic Instability
o Once microtubule nucleated, it typically grows outward from the organizing center by addition of alpha beta tubulin dimers to
its plus end (and can suddenly shrink)
o Switching back and forth between polymerization and depolymerization = dynamic instability
o Centrosome is continually shooting out new microtubules in different directions in an exploratory fashion, many of which
retract
o Can be prevented from disassembly if its plus end is stabilized by attachment to another molecule or cell structure so as to
prevent its depolymerization
o If stabilized by attachment to a structure in a more distant region of the cell, the microtubule will establish a relatively stable
link between that structure of centrosome
- Dynamic Instability is Driven by GTP Hydrolysis
o Due to intrinsic capacity of tubulin dimers to hydrolyze GTP
o Each free tubulin dimer has one GTP molecule bound to beta tubulin, which hydrolyzes GTP to GDP after the dimer added to
growing microtubule. This GDP remains bound to the beta tubulin
o During rapid polymerization, tubulin dimers add to end faster than the GTP is hydrolyzed rapidly growing end composed
entirely of GTP tubulin dimers that forms GTP cap
o GDP tubulin that is freed as microtubule depolymerizes joins pool of unpolymerized tubulin in cytosol, forming a pool of
tubulin dimers available for microtubule growth
o Tubulin dimers joining the pool rapidly exchange bound GDP for GTP, becoming competent again to add another microtubule
that is in its growth phase
- Microtubule Dynamics Can be Modified by Drugs
o If a cell in mitosis is exposed to the drug colchicine, which binds tightly to free tubulin dimers and prevents their
polymerization into microtubules, the mitotic spindle rapidly disappears, and the cell stalls in the middle of mitosis, unable to
partition the chromosomes into two groups.
o Demonstrates that mitotic spindle is normally maintained by a continuous balanced addition and loss of tubulin subunits:
when tubulin addition is blocked by colchicine, tubulin loss continues until the spindle disappears
o Drug Taxol (opposite effect) binds to microtubules and prevents from losing subunits
o Inactivation or destruction of mitotic spindle kills dividing cells
- Microtubules Organize the Cell Interior
o As cells enter mitosis, microtubules become initially more dynamic, switching between
growing and shrinking more frequently than cytoplasmic microtubules normally do
o Stabilized microtubules serve to maintain organization of a differentiated cell
o Most differentiated animal cells are polarized
o Polarity helps position organelles in their required location within the cell and to guide streams of vesicular and
macromolecular traffic moving throughout the cell
o Activity of microtubules depends on large variety of accessory proteins that bind to them
o Some of these microtubule-associated proteins stabilize microtubules against disassembly or motor proteins actively transport
- Motor Proteins Drive Intracellular Transport
o Saltatory movement of membrane-enclosed organelles and vesicles is much more
sustained and directional than the continual, small, Brownian movements caused by random thermal motions – occur along
either microtubules or actin filaments
o Movement driven by motor proteins use energy derived from repeated cycles of ATP hydrolysis to travel steadily along the
microtubule or actin filament in a single direction
o Motor proteins attach to other cell components transport cargo along filaments
o Motor proteins that move along cytoplasmic microtubules belong to 2 families: the kinesins move toward plus end of
microtubule (outward from cell body); the dyneins move toward minus end (toward cell body)
o Both are dimers that have 2 globular ATP-binding heads and single tail. The heads interact with microtubules is stereospecific
manner. The tail binds stably to some cell component which determines type of cargo that motor protein can transport
o Globular heads are enzymes with ATP hydrolyzing activity (ATPase) that provides energy for driving a directed series of
conformational changes in the head that enable it to move along microtubule
- Microtubules and Motor Proteins Position Organelles in the Cytoplasm
o ER extends out from point of connection with nuclear envelope along microtubules, which reach from the centrally located
centrosome out to the PM
o As cell grows, kinesins attached to outside the ER membrane (via receptor proteins) pull the ER outward along microtubules
stretching it like a net
o Cytoplasmic dyneins attached to golgi membrane pulls golgi along microtubules in opposite direction, inward toward nucleus
o Colchicine changes location of ER and golgi
- Cilia and Flagella Contain Stable Microtubules Moved by Dynein
o Cells use stable microtubules as support in construction of polarized structures
o Cilia covered by plasma membrane that extend from surface of cells; each containing a core of stable microtubules, arranged
in a bundle that grow from a cytoplasmic basal body, which serves as an organizing center
o Flagella are designed to move entire cell, rather than moving fluid across cell surface; propagate waves along their length,
propelling the attached cell along
o Cross section of cilium shows 9 doublet microtubules arranged arranged in a ring around a pair of single microtubules (9+2
array characteristic to flagella and cilia)
o Movement for both produced by bending of core as microtubules slid against each other.
The microtubules are associated with numerous accessory proteins, which project at regular positions along the length of the
microtubule bundle. Some proteins serve as cross-links to hold the bundle of microtubules together; others generate force that
causes cilium to bend
o Most important of accessory protein is motor protein ciliary dynein that generates bending motion of core
Actin Filaments
- Actin filaments, polymers of the protein actin, are present in all eukaryotic cells and are essential for many of the cell’s
movements, especially those involving the cell surface
- Like microtubules, many actin filaments are unstable, but by associating with other proteins they can also form stable
structures in cells, such as contractile apparatus of muscle cells
- Interact with large number of actin-binding proteins that enable the filaments to serve a variety of function in cells
- Depending on which protein they associate with, actin filaments can form stiff and stable structures, such as microvilli on
epithelial cells or small contractile bundles that can contract and act like tiny muscles in animal cells
- Can also form temporary structures, such as dynamic protrusions formed at the leading edge of a crawling cell or the
contractile ring that pinches cytoplasm in 2 when animal cells divide
- Actin-dependent movements usually require actin’s association with a motor protein = myosin
- Actin Filaments Are Thin and Flexible
o Each filament is a twisted chain of identical globular actin monomers, all of which “point” in same direction along the axis of
the chain. Like a microtubule, an actin filament has a structural polarity, with a plus end and a minus end
o Actin filaments are thinner, more flexible, and usually shorter than microtubules, but many more of them
o Unlike intermediate filaments and microtubules, rarely occur in isolation – generally found in cross-linked structures, which
are much stronger than individual filaments
- Actin and Tubulin Polymerize by Similar Mechanisms
o Actin filaments grow by addition of actin monomers, faster at plus end than minus
o Naked actin filament unstable, and can disassemble from both ends
o Free actin monomers carry ATP hydrolyzing it to ADP soon after incorporation, reducing the strength of binding between
monomers and decreased stability of the polymer
o At intermediate concentrations of free actin, however, something interesting takes place.
Actin monomers add to the plus end at a rate faster than the bound ATP can be hydrolyzed, so the plus end grows. At the minus
end, by contrast, ATP is hydrolyzed faster than new monomers can be added; because ADP-actin destabilizes the structure, the
filament loses subunits from its minus end at the same time as it adds them to the plus end.
o Treadmilling of actin filaments and dynamic instability of microtubules rely on hydrolysis of a bound nucleoside triphosphate
to regulate length of polymer
o Treadmilling – simultaneous gain of monomers at plus end and loss at minus
o Dynamic instability – rapid switch from growth to shrinkage (or reverse) at only plus end
o Microtubules undergo more drastic changes in length than do filaments
- Many Proteins Bind to Actin and Modify Its Properties
o Unlike tubulin dimers, concentration of actin monomers is high
o Cells contain small proteins (like thymosin and profilin) that bind to actin monomers in cytosol, preventing them from adding
to the ends of actin filaments
o When actin filaments needed, other actin-binding proteins such as formins and actin-related proteins (ARPs) promote actin
polymerization
o There are many actin-binding proteins that mostly bind to assembled actin filaments and control behavior of intact filaments
- A Cortex Rich in Actin Filaments Underlies the Plasma Membrane of Most Eukaryotic Cells
o Although actin is found throughout the cytoplasm of a eukaryotic cell, in most cells it is highly concentrated in a layer just
beneath the PM. In this region, called the cell cortex, actin filaments are linked by actin-binding proteins into a meshwork that
supports the plasma membrane and gives it mechanical strength
o These filaments become cross-linked into a three-dimensional meshwork, which governs cell shape and the mechanical
properties of the plasma membrane: the rearrangements of actin filaments within the cortex provide much of the molecular
basis for changes in both cell shape and cell locomotion
- Cell Crawling Depends on Cortical Actin
o Chemotactic molecules binding to receptors on the cell surface triggers changes in actin filament assembly that help direct the
cells toward their prey
o Three interrelated essential processes: (1) the cell pushed out protrusions at its “front” or leading edge; (2) these protrusions
adhere to the surface over which the cell is crawling; and (3) the rest of the cell drags itself forward by traction on these
anchorage points
o First step driven by actin polymerization: leading edge of a crawling fibroblast in culture regularly extends thin, sheetlike
lamellipodia, which contain dense network of actin filaments, oriented so that most of the filaments have their plus ends close
to PM
o Many cells also extend thin, stiff protrusions called filopodia at leading edge and surface
o Both lamellipodia and filopodia are exploratory, motile structures that form and retract with great speed and thought to be
generated by rapid local growth of actin filaments, which assemble clos to PM and elongate by addition of actin monomers at
their plus ends
o The formation and growth of actin filaments at the leading edge of a cell are assisted by various actin-binding proteins. The
actin-related proteins (ARPs) promote formation of a web of branched actin filaments in lamellipodia. ARPs form complexes that
bind to sides of existing actin filaments and nucleate formation of new filaments, that grow out at an angle to make side
branches
o The other kind of cell protrusion, the filopodium, depends on formins, a nucleating protein that attaches to the growing plus
ends of actin fila- ments and promotes the addition of new monomers to form straight, unbranched filaments. Formins are also
used elsewhere to assemble unbranched filaments, as in the contractile ring that pinches a dividing animal cell in two
- Actin Associates with Myosin to Form Contractile Structures
o All actin-dependent motor proteins belong to the myosin family. They bind to and hydrolyze ATP, which provides the energy
for their movement along actin filaments toward the plus end.
o Myosin-I molecules have a head domain and a tail. The head domain binds to an actin filament and has the ATP-hydrolyzing
motor activity that enables it to move along the filament in a repetitive cycle of binding, detachment, and rebinding. The tail
varies among the different types of myosin-I and determines what type of cargo the myosin drags along.
- Extracellular Signals Can Alter the Arrangement of Actin Filaments
o Extracellular signal molecules that regulate actin cytoskeleton activate many cell-surface receptor proteins, which activate
many intracellular signaling pathways. These pathways often converge on a group of closely related monomeric GTPase proteins
called the Rho protein family.
o Activation of different members of Rho family affects organization of actin filaments indifferent ways
o These structural changes occur because the Rho family GTP-binding proteins, together with the protein kinases and accessory
proteins with which they interact, act like a computational network to control actin organization and dynamics.
o This network receives external signals from nutrients, growth factors, and contacts with neighboring cells and the extracellular
matrix, along with intracellular information about the cell’s metabolic state and readiness for division.
o The Rho network then processes these inputs and activates intracellular signaling pathways that shape the actin cytoskeleton
Muscle Contraction
- Muscle Contraction Depends on Interacting Filaments of Actin and Myosin
o Muscle myosin belongs to the myosin-II subfamily of myosins, all of which are dimers, with 2 globular ATPase heads at one end
and 1 coiled-coil tail at the other
o Clusters of myosin-II molecules bind to each other through their coiled-coil tails, forming a bipolar myosin filament from which
the heads project
o The myosin filament has 2 sets of myosin heads pointing in opposite directions, away from the middle. One set binds to actin
filaments in one orientation and moves the filaments one way; the other set binds to other actin filaments in the opposite
orientation and moves the filaments in the opposite direction. As a result, a myosin filament slides sets of oppositely oriented
actin filaments past one another contractile force
- Actin Filaments Slide Against Myosin Filaments During Muscle Contraction
o Skeletal muscle fibers are formed by the fusion of many separate smaller cells
o Nuclei of cells are retained in muscle fiber and lie just beneath the PM
o Bulk of cytoplasm made up of myofibrils, the contractile elements of muscle cell
o A myofibril consists of a chain of identical tiny contractile units, or sarcomeres – highly organized assemblies of 2 types of
filaments: actin and myosin composed of a muscle specific form of myosin-II
o The myosin filaments (thick) are centrally positioned in each sarcomere, whereas the slender actin filaments (thin) extend
inward from each end of sarcomere, where they are anchored by their plus end to the Z disc (minus end overlap ends of myosin
filaments)
o Contraction of muscle cell caused by simultaneous shortening of a cell’s sarcomeres, caused by actin filaments sliding past
myosin filaments, with no change in length of either. The sliding motion made by myosin heads that project from sides of
myosin filament and interact with adjacent actin filaments.
o When muscle contracts, myosin heads start to walk along actin filament in repeated cycles of attachment and detachment
(myosin head binds to hydrolyze one molecule of ATP conformational changes that moves tip of head along actin toward plus
end)
o Myosin head pulls against actin filament causing it to slide against myosin filament
o All sarcomeres of muscle coupled together triggered simultaneously by signaling system
- Muscle Contraction is Triggered by a Sudden Rise in Cytosolic Ca2+
o NT released from nerve terminal triggers an action potential in muscle cell PM
o Electrical excitation spreads into series of membranous tubes, called transverse (or T) tubules, that extend inward from PM
around each myofibril
o Electrical signal relayed to sarcoplasmic reticulum, an adjacent sheath of interconnected
flattened vesicles that surrounds each myofibril that is a specialized region of ER in muscle cells; contains high concentration of
Ca2+
o In response to electrical excitation, much of this Ca2+ is released into cytosol through ion channels that open in sarcoplasmic
reticulum membrane
o In muscle, rise in cytoplasmic Ca2+ activates a molecular switch made of specialized accessory proteins (like tropomyosin)
closely associated with actin filaments
o Tropomyosin binds in groove of actin helix, where it prevents myosin heads from associating with actin filament
o Troponin - protein complex with Ca2+ sensitive proteins at end of tropomyosin
o When increase in Ca2+ in cytosol, Ca2+ binds to troponin and induces a change in shape of complex that causes tropomyosin
molecules to shift positions, allowing myosin heads to bind to actin filaments and initiate contraction
Chapter 18 – The Cell-Division Cycle
Lecture 19
- A cell reproduces by carrying out orderly sequence of events in which it duplicates its contents and then divides in 2 cell cycle
Overview of the Cell Cycle
- To maintain their size, dividing cells coordinate their growth and division
- The Eukaryotic Cell Cycle Usually Includes Four Phases
o Mitosis – nucleus divides; cytokinesis – cell splits in two
o These two processes = M phase of the cycle
o The period between one M phase and the next is called interphase (where the cell simply increases in size, encompasses the
remaining 3 phases of the cell cycle)
o S phase (S=synthesis) – cell replicates its DNA and is flanked by two “gap” phases – G1 and G2 – during which the cell
continues to grow
o During these gap phases, cell monitors internal and external environment to ensure suitable conditions before the cell
commits to major upheavals of S phase (which follows G1) and mitosis (following G2)
o During all of interphase, a cell continues to transcribe genes, synthesize proteins, and grow in mass. Together with S phase, G1
and G2 provide time needed for cell to grow and duplicate its cytoplasmic organelles
- A Cell-Cycle Control System Triggers the Major Processes of the Cell Cycle
o Eukaryotic cells possess complex network of regulatory proteins known as the cell-cycle control system that guarantees that
the events of the cell cycle – DNA replication, mitosis, so on – occur in a set sequence and that each process has been completed
before the next begins
o Cell-cycle control system achieved by employing molecular breaks, called checkpoints, to pause the cycle at certain transition
points so as to not trigger the next step unless the cell is properly prepared
o 3 main transition points
o At transition from G1 to S phase, the control system confirms that the environment is favorable for proliferation before
committing to DNA replication – requires both sufficient nutrients and specific signal molecules in extracellular environment
(Go)
o At transition from G2 to M phase, the control system confirms that the DNA is undamaged and fully replicated
o During mitosis, ensures that duplicated chromosomes are properly attached to a cytoskeletal machine called the mitotic
spindle before spindle pulls chromosomes apart
- Cell-Cycle Control is Similar in All Eukaryotes
The Cell-Cycle Control System
- The cell-cycle control system switches machinery on and off at correct times
- Core of cell-cycle control system is a series of molecular switches that operate in a defined sequence and orchestrate main
events of cycle, including DNA replication/segregation
- The Cell-Cycle Control System Depends on Cyclically Activated Protein Kinases called Cdks
o Regulation carried out largely through phosphorylation (specific set of protein kinases) and dephosphorylation (set of protein
phosphatases) of proteins involved
o Switching kinases on and off at appropriate times due to another set of proteins in the control system – the cyclins that need
to bind to cell-cycle kinases before the kinases can become enzymatically active
o Kinases are known as cyclin-dependent protein kinases, or Cdks
o Cyclical changes in cyclin concentrations help drive the cyclic assembly and activation of cyclin-Cdk complexes that once
activated help trigger cell-cycle events
- Different Cyclin-Cdk Complexes Trigger Different Steps in the Cell Cycle
o The cyclin that acts on G2 to trigger entry into M phase is called M cyclin, and the active complex it forms with Cdk is called MCdk
o Other cyclins, called S cyclins and G1/S cyclins, bind to a distinct Cdk protein late in G1 to form S-Cdk and G1/S-Cdk that help
launch the S phase
o Another group of cyclins, called G1 cyclins, act earlier in G1 and bind to other Cdk proteins to form G1-Cdks, which help drive
the cell through G1 toward S phase
o Each of these cyclin-Cdk complexes phosphorylates a different set of target proteins in the cell that triggers different transition
steps in the cycle
- Cyclin Concentrations are Regulated by Transcription and by Proteolysis
o Over course of cell cycle, the concentration of each type of cyclin rises gradually and then falls abruptly
o Increase stems from increasing transcription of cyclin genes, whereas the rapid fall is precipitated by a full-scale targeted
destruction of the protein
o Degradation of M and S cyclins part way through M phase depends on large enzyme complex called anaphase-promoting
complex (APC) that tags cyclins with a chain of ubiquitin (proteins marked this way are directed to proteasomes for degradation)
o Ubiquitylation and degradation of cyclin returns its Cdk to inactive state
o Cyclin destruction can help drive transition from one phase of cycle to next
- The Activity of Cyclin-Cdk Complexes Depends on Phosphorylation and Dephosphorylation
o Cyclin-Cdk complex contains inhibitory phosphatases, and to become active, the Cdk must be dephosphorylated by a specific
protein phosphatase
o Thus, protein kinases and phosphatases regulate activity of specific cyclin-Cdk complexes and help control progression through
cell cycle
- Cdk Activity Can be Blocked by Cdk Inhibitor Proteins
o Activity of Cdks can also be modulated by the binding of Cdk inhibitor proteins to block the assembly or activity of certain
cyclin-Cdk complexes
o Some help maintain Cdks in an inactive state during G1 phase, delaying progression inro S phase to give cell more time to grow
or wait for favorable extracellular conditions
- The Cell-Cycle Control System Can Pause the Cycle in Various Ways
o Allow for entry into S phase only if environmental conditions are appropriate
o Triggers mitosis only after DNA has been completely replicated
o Initiates chromosome segregation only after duplicated chromosomes are correctly aligned at mitotic spindle
o G1-to-S transition: uses Cdk inhibitors to keep cells from entering S and replicating DNA
o G2-to-M transition: suppresses activation of M-Cdk by inhibiting phosphatase needed to activate the Cdk
o Can delay the exit from mitosis by inhibiting activation of APC, preventing degradation of M cyclin
G1 Phase
- Period of metabolic activity, cell growth, and repair
- Based on intracellular signals about size of the cell and extracellular signals about environment
- Cell-cycle control machinery can either hold the cell transiently in G1 or allow it to enter S phase
- Once past critical G1-to-S transition, a cell usually continues through rest quickly
- Cdks are Stably Inactivated in G1
o To usher a cell M phase to G1, the cell-cycle control machinery must inactivate its inventory of S-Cdk and M-Cdk by eliminating
all of the existing cyclins and blocking the synthesis of new ones, and mu deploying Cdk inhibitor proteins to muffle activity of
any remaining cyclin-Cdk complexes
o Ensuring that essentially all Cdk activity is shut down
- Mitogens Promote the Production of the Cyclins that Stimulate Cell Division
o Mammalian cells multiply only if stimulated to do so by extracellular signals, called mitogens, produced by other cells
o Escape from cell-cycle arrest – or from certain nonproliferating states – requires accumulation of cyclins
o Mitogens switch on cell signaling pathways that stimulate the synthesis of G1 cyclins, G1/S cyclins, and other proteins involved
in DNA synthesis and chromosome duplication
o Buildup of cyclins wave of G1/S-Cdk activity that relieves negative controls that block progression from G1 to S phase
o Crucial negative control mediated by Retinoblastoma (Rb) protein that binds to transcription regulators to prevent them from
turning on the genes required for cell prolif.
o Mitogens release Rb brake by triggering activation of G1-Cdks and G1/S-Cdks by phosphorylating the Rb protein, altering its
conformation so that it releases its bound transcription regulators, which are then free to activate genes for cell proliferation
- DNA Damage Can Temporarily Halt Progression Through G1
o Mechanism that operates at G1-to-S transition - prevents cell replicating damaged DNA
o DNA damage in G1 causes increase in concentration and activity of a protein called p53, which is a transcription regulator that
activates the transcription of a gene encoding a Cdk
inhibitor protein called p21
o P21 binds to G1/S-Cdk and S-Cdk, preventing them from driving cell into S phase
o If DNA damage too severe to repair, p53 can induce the cell to kill itself through apoptosis
o Mutations in p53 gene are found in about half of all human cancers
- Cells Can Delay Division for Prolonged Period by Entering Specialized Nondividing States
o Can withdraw from cell cycle for prolonged periods – temporarily or permanently
o In terminally differentiated cells, the cell-cycle control system is dismantled completely and genes encoding the relevant
cyclins and Cdks are irreversibly shut down
o In absence of appropriate signals, other cell type withdraw temporarily, entering arrested state called G0 (Go)
o Most liver cells in Go, but can be stimulated if liver is damaged
S Phase
- Initiating DNA replication and preventing replication from happening more than once per cell
- S-Cdk Initiates DNA Replication and Blocks Re-Replication
o Early in G1, DNA is made replication ready by recruitment of proteins to the sites along each chromosome where replication
will begin
o The origin recognition complex (ORC) recruits a protein called Cdc6 and together they load the DNA helicases that will open
double helix and ready the origin of replication
o Signal to commence replication comes from S-Cdk, the cyclin-Cdk complex that triggers S phase and becomes
assembled/activated at end of G1
o During S phase it activates DNA helicases in prereplicative complex and promotes assembly of the rest of the proteins that
form the replication fork
o S-Cdk triggers initiation of DNA synthesis and prevents re-replication by helping phosphorylate Cdc6, which marks protein for
degradation
- Incomplete Replication Can Arrest the Cell Cycle in G2
o Cell-cycle control system can delay entry into M phase
o M-Cdk activity is inhibited by phosphorylation at particular sites. These must be removed by an activating protein phosphatase
called Cdc25
o When DNA damaged or incompletely replicated, Cdc25 is inhibited, preventing the removal of inhibitory phosphatases M-Cdk
remaining inactive and M phase delayed
M Phase
- M phase = mitosis + cytokinesis
- Cell reorganizes virtually all of its components and distributes them equally into the two daughter cells
- M-Cdk Drives Entry into M Phase and Mitosis
o M-Cdk helps prepare the duplicated chromosomes for segregation and induces the assembly of the mitotic spindle – the
machinery that pulls duplicated chromosomes apart
o M-Cdk complexes accumulate throughout G2 but not activated until end of G2, when activating phosphatase Cdc25 removes
inhibitory phosphates holding M-Cdk activity in check
o Once activated, each M-Cdk complex can indirectly activate additional M-Cdk complexes – by phosphorylating and activating
more Cdc25
o Overall consequence: once M-Cdk activation begins, it ignites an explosive increase in M-Cdk activity that drives the cell
abruptly from G2 into M phase
- Cohesins and Condensins Help Configure Duplicated Chromosomes for Separation
o Protein complexes called condensins help carry out chromosome condensation, which reduces mitotic chromosomes to
compact bodies that can be more easily segregated within the crowded confines of the cell
o Assembly of condensing complexes onto the DNA is triggered by phosphorylation of condensins by M-Cdk
o Identical copies called sister chromatids each contain a single, double-stranded molecule of DNA, along with its associated
proteins held together by protein complexes called cohesins
o Cohesins and condensins structurally related, both thought to form ring structures around chromosomal DNA
o Condensins assemble on each individual sister chromatid at the start of the M phase and help each of these double helices to
coil up into a more compact form
- Different Cytoskeletal Assemblies Carry Out Mitosis and Cytokinesis
o Mitotic spindle carries out nuclear division (mitosis)
o Contractile ring carries out cytoplasmic division (cytokinesis)
o Mitotic spindle is composed of microtubules and the various proteins that interact with them
o The contractile ring consists mainly of actin filaments and myosin filaments arranged in a ring around equator of cell and starts
to assemble beneath PM toward end of mitosis
- M Phase Occurs in Stages
o First five phases of M phase – prophase, prometaphase, metaphase, anaphase, and telophase – constitute mitosis
o Cytokinesis, which constitutes the final stage of M phase, begins before mitosis ends
Mitosis
- During mitosis, cohesion proteins are cleaved, sister chromatids split apart, and resulting daughter chromosomes are pulled to
opposite poles of the cell by the mitotic spindle
- Centrosomes Duplicate to Help form the Two Poles of the Mitotic Spindle
o Before M phase begins DNA must be fully replicated and centrosome must be duplicated
o The centrosome is the principle microtubule-organizing center in animal cells that duplicates so it can help form the 2 poles of
the mitotic spindle so that each daughter cell can receive its own centrosome
o Centrosome duplication begins at same time as DNA replication and is triggered by same Cdks – G1/S-Cdk and S-Cdk – that
initiate DNA replication
o Two centrosomes separate and each nucleates a radial array of microtubules called an aster that move to opposite ends of the
nucleus to form the two poles of the mitotic spindle
o Process of centrosome duplication and separation is called centrosome cycle
- The Mitotic Spindle Starts to Assemble in Prophase
o Mitotic spindle begins to form in prophase
o At start of mitosis stability of microtubules decreases – in part because M-Cdk phosphorylates microtubule-associated proteins
that influence the stability of the microtubules
o During prophase, rapidly growing and shrinking microtubules extend in all directions from the two centrosomes, exploring the
interior of the cell.
o Interaction between microtubules growing from one centrosome with microtubules from the other stabilizes the
microtubules, preventing them from depolymerizing, and it joins the two sets of microtubules together to form the basic
framework of the mitotic spindle, with its characteristic bipolar shape
o The two centrosomes that give rise to these microtubules are now called spindle poles, and the interacting microtubules are
called interpolar microtubules. The assembly of the spindle is driven, in part, by motor proteins associated with the interpolar
microtubules that help to cross-link the two sets of microtubules.
- Chromosomes Attach to the Mitotic Spindle at Prometaphase
o Prometaphase starts abruptly with the disassembly of the nuclear envelope, which breaks up into small membrane vesicles.
This process is triggered by the phosphorylation and consequent disassembly of nuclear pore proteins and the intermediate
filament proteins of the nuclear lamina, the network of fibrous proteins that underlies and stabilizes the nuclear envelope
o The spindle microtubules grab hold of the chromosomes at kinetochores, protein complexes that assemble on the centromere
of each condensed chromosome during late prophase
o Kinetochores recognize the special DNA sequence present at the centromere: if this sequence is altered, kinetochores fail to
assemble and, consequently, the chromosomes fail to segregate properly during mitosis.
o Because kinetochores on sister chromatids face in opposite directions, they tend to attach to microtubules from opposite
poles of the spindle, so that each duplicated chromosome becomes linked to both spindle poles. The attachment to opposite
poles, called bi-orientation, generates tension on the kinetochores, which are being pulled in opposite directions. This tension
signals to the sister kinetochores that they are attached correctly and are ready to be separated
- Chromosomes Assist in the Assembly of the Mitotic Spindle
o In cells without centrosomes—including all plant cells and some animal cell types—the chromosomes nucleate microtubule
assembly, and motor proteins then move and arrange the microtubules and chromosomes into a bipolar spindle. Even in animal cells that normally have centrosomes, a bipolar spindle can still be formed in this way if the centrosomes are removed
- Chromosomes Line Up at the Spindle Equator at Metaphase
o They align at the equator of the spindle, halfway between the two spindle poles, thereby forming the metaphase plate. This
event defines the beginning of metaphase
o A continuous balanced addition and loss of tubulin subunits is also required to maintain the metaphase spindle: when tubulin
addition to the ends of microtubules is blocked by the drug colchicine, tubulin loss continues until the metaphase spindle
disappears.
o the duplicated chromosomes are not simply deposited at the metaphase plate. They are suspended there under tension. In
anaphase, that tension will pull the sister chromatids apart.
- Proteolysis Triggers Sister-Chromatid Separation at Anaphase
o Anaphase begins abruptly with the breakage of the cohesin linkages that hold sister chromatids together
o This release allows each chromatid—now considered a chromosome—to be pulled to the spindle pole to which it is attached
o The cohesin linkage is destroyed by a protease called separase. Before anaphase begins, this protease is held in an inactive
state by an inhibitory protein called securin. At the beginning of anaphase, securin is targeted for destruction by APC—the same
protein complex, discussed earlier, that marks M cyclin for degradation. Once securin has been removed, separ- ase is then free
to sever the cohesin linkages
- Chromosomes Segregate During Anaphase
o In anaphase A, the kinetochore microtubules shorten and the attached chromosomes move poleward. In anaphase B, the
spindle poles themselves move apart, further segregating the two sets of chromosomes
o The driving force for the movements of anaphase A is thought to be provided mainly by the loss of tubulin subunits from both
ends of the kinetochore microtubules. The driving forces in anaphase B are thought to be provided by two sets of motor
proteins—members of the kinesin and dynein families—operating on different types of spindle microtubules
- An Unattached Chromosome Will Prevent Sister-Chromatid Separation
o To monitor chromosome attachment, the cell makes use of a negative signal: unattached chromosomes send a “stop” signal to
the cell-cycle control system. Although only some of the details are known, the signal inhibits further progress through mitosis
by blocking the activation of the APC. Without active APC, the sister chromatids remain glued together. Thus, none of the
duplicated chromosomes can be pulled apart until every chromosome has been positioned correctly on the mitotic spindle. This
so-called spindle assembly checkpoint thereby controls the onset of anaphase, as well as the exit from mitosis, as mentioned
earlier
- The Nuclear Envelope Re-forms at Telophase
o During telophase, the final stage of mitosis, the mitotic spindle disassembles, and a nuclear envelope reassembles around each
group of chromosomes to form the two daughter nuclei
o the nuclear pore proteins and nuclear lamins that were phosphorylated during pro- metaphase are now dephosphorylated,
which allows them to reassemble and the nuclear envelope and lamina to re-form
o Once the nuclear envelope has re-formed, the pores pump in nuclear proteins, the nucleus expands, and the condensed
chromosomes decondense into their interphase state. As a consequence of this decondensation, gene transcription is able to
resume. A new nucleus has been created, and mitosis is complete.
Cytokinesis
- Cytokinesis, the process by which the cytoplasm is cleaved in two, completes M phase.
- cytokinesis in animal cells depends on a transient structure based on actin and myosin filaments, the contractile ring
- Both the plane of cleavage and the timing of cytokinesis are determined by the mitotic spindle.
- The Mitotic Spindle Determines the Plane of Cytoplasmic Cleavage
o This positioning ensures that the cleavage furrow cuts between the two groups of segregated chromosomes, so that each
daughter cell receives an identical and complete set of chromosomes.
o during anaphase, the overlapping interpolar microtubules that form the central spindle recruit and activate proteins that
signal to the cell cortex to initiate the assembly of the contractile ring at a position midway between the spindle poles.
o In most of these asymmetric divisions, the daughters also differ in the molecules they
inherit, and they usually develop into different cell types.
- The Contractile Ring of Animal Cells is Made of Actin and Myosin Filaments
o The contractile ring is composed mainly of an overlapping array of actin filaments and myosin filaments. It assembles at
anaphase and is attached to membrane-associated proteins on the cytoplasmic face of the plasma membrane. Once assembled,
the contractile ring is capable of exerting a force strong enough to bend a fine glass needle inserted into the cell before
cytokinesis. Much of this force is generated by the sliding of the actin filaments against the myosin filaments.
o Contractile ring is a transient structure: it assembles to carry out cytokinesis, gradually becomes smaller as cytokinesis
progresses, and disassembles completely once the cell has been cleaved in two.
- Cytokinesis in Plant Cells Involves Formation of a New Cell Wall
o The new cell wall starts to assemble in the cytoplasm between the two sets of segregated chromosomes at the start of
telophase. The assembly process is guided by a structure called the phragmoplast, which is formed by the remains of the
interpolar microtubules at the equator of the old mitotic spindle.
o Small membrane-enclosed vesicles, largely derived from the Golgi apparatus and filled
with polysaccharides and glycoproteins required for the cell-wall matrix, are transported along the microtubules to the
phragmoplast. Here, they fuse to form a disclike, membrane-enclosed structure, which expands outward by further vesicle
fusion until it reaches the plasma membrane and original cell wall, thereby dividing the cell in two.
o Later, cellulose microfibrils are laid down within the matrix to complete the construction of the new cell wall.
- Membrane-Enclosed Organelles Must be Distributed to Daughter Cells When a Cell Divides
o Upon entry into M phase, the reorganization of the microtubules releases the ER; in most cells, the released ER remains intact
during mitosis and is cut in two during cytokinesis.
The Golgi apparatus fragments during mitosis; the fragments associate with the spindle microtubules via motor proteins,
thereby hitching a ride into the daughter cells as the spindle elongates in anaphase
o Other components of the cell – including the other membrane-enclosed organelles, ribosomes, and all of the soluble proteins
– are inherited randomly when a cell divides
Control of Cell Numbers and Cell Size
- 3 fundamental processes determine organ and body size: cell growth, cell division, and cell death.
- Apoptosis Helps Regulate Animal Cell Numbers
o If cells are no longer needed, they can commit suicide by activating an intracellular death program—a process called
programmed cell death. In animals, the most common form of programmed cell death is called apoptosis
- Apoptosis is Mediated by an Intracellular Proteolytic Cascade
o Cells that die as a result of acute injury typically swell and burst, spilling their contents all over their neighbors, a process called
cell necrosis. This eruption triggers a potentially damaging inflammatory response.
o a cell that undergoes apoptosis dies neatly, without damaging its neighbors
o Most importantly, the cell surface is altered in such a manner that it immediately attracts phagocytic cells, usually specialized
phagocytic cells called macrophages. These cells engulf the apoptotic cell before it spills its contents
o The molecular machinery responsible for apoptosis, which seems to be similar in most animal cells, involves a family of
proteases called caspases. These enzymes are made as inactive precursors, called procaspases, which are activated in response
to signals that
induce apoptosis
o Initiator caspases cleave, and thereby activate, downstream executioner caspases. Some of these executioner caspases then
activate additional executioners, kicking off an amplifying, proteolytic cascade; others dismember other key proteins in the cell
o Irreversible
- The Intrinsic Apoptotic Death Program is Regulated by the Bcl2 Family of Intracellular
Proteins
o The main proteins that regulate the activation of caspases are members of the Bcl2 family of intracellular proteins
o Two of the most important death-inducing family members are proteins called Bax and Bak. These proteins—which are
activated in response to DNA damage or other insults—promote cell death by inducing the release of the electron-transport
protein cytochrome c
from mitochondria into the cytosol. Other members of the Bcl2 family (including Bcl2 itself) inhibit apoptosis by preventing Bax
and Bak from releasing cytochrome c. The
balance between the activities of pro-apoptotic and anti-apoptotic members of the Bcl2 family largely determines whether a
mammalian cell lives or dies by apoptosis.
o The cytochrome c molecules released from mitochondria activate initiator procaspases—and induce cell death—by promoting
the assembly of protein complex called an apoptosome.
o The apoptosome then recruits and activates a particular initiator pro- caspase, which then triggers a caspase cascade that
leads to apoptosis
- Extracellular Signals Can Also Induce Apoptosis
o Others stimulate apoptosis more directly by activating a set of cell-surface receptor proteins known as death receptors
o Fas is activated by a membrane-bound protein, called Fas ligand, present on the surface of specialized immune cells called
killer lymphocytes. These killer cells help regulate immune responses by inducing apoptosis in other immune cells that are
unwanted or are no longer needed
- Animal Cells Require Extracellular Signals to Survive, Grow, and Divide
o Most of the extracellular signal molecules that influence cell survival, cell growth, and cell division are either soluble proteins
secreted by other cells or proteins that are bound to the surface of other cells or to the extracellular matrix.
o The positively acting signal proteins can be classified, on the basis of their function, into three major categories:
Survival factors promote cell survival, largely by suppressing apoptosis.
Mitogens stimulate cell division, primarily by overcoming the intracellular braking mechanisms that tend to block progression
through the cell cycle.
Growth factors stimulate cell growth (an increase in cell size and mass) by promoting the synthesis and inhibiting the
degradation of proteins and other macromolecules.
- Survival Factors Suppress Apoptosis
o If deprived of such survival factors, cells activate a caspase-dependent intracellular suicide program and die by apoptosis.
o Survival factors usually act by activating cell-surface receptors. Once activated, the
receptors turn on intracellular signaling pathways that keep the apoptotic death program suppressed, usually by regulating
members of the Bcl2 family of proteins. Some survival factors, for example, increase the production of Bcl2, a protein that
suppresses apoptosis
- Mitogens Stimulate Cell Division by Promoting Entry into S Phase
o One of the first mitogens identified in this way was platelet-derived growth factor, or PDGF, the effects of which are typical of
many others discovered since. When blood clots form (in a wound, for example), blood platelets incorporated in the clots are
stimulated to release PDGF. PDGF then binds to receptor tyrosine kinases in surviving cells at the wound site, stimulating the
cells to proliferate and help heal the wound.
Similarly, if part of the liver is lost through surgery or acute injury, a mitogen called hepatocyte growth factor helps stimulate the
surviving liver cells to proliferate.
- Growth Factors Stimulate Cells to Grow
o Like most survival factors and mitogens, most extracellular growth fac- tors bind to cell-surface receptors that activate
intracellular signaling pathways. These pathways lead to
the accumulation of proteins and other macromolecules. Growth factors both increase the rate of synthesis of these molecules
and decrease their rate of degradation
- Some Extracellular Signal Proteins Inhibit Cell Survival, Division, or Growth
o Myostatin, for example, is a secreted signal protein that normally inhibits the growth and proliferation of the precursor cells
(myoblasts) that fuse to form skeletal muscle cells during mammalian development
o Because cancer cells are generally less dependent than normal cells on signals from other cells, they can out-survive, outgrow,
and out- divide their normal neighbors, producing tumors that can kill their host