Molecules and Cells notes
Intro to cells
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Cells can be solitary or part of a community
Individual cells of multicellular organisms are specialized and have intricate functions and forms
and communications
Cells are broken down into prokaryotes and eukaryotes
Prokaryotes
o Can be many shapes
 Spherical
 Rod
 Corkscrew/spiral
o Reproduces quickly (about 20 min)
o Most diverse cells
 Explore many habitats that eukaryotes cannot tolerate
o Broken down into bacteria and archaea
 The archaea are the prokaryotic cells that live in extreme places (extremophiles)
Eukaryotic cells
o Larger defined membrane bound organelles with different functions
o Cytoskeleton that directs movement
Model organisms
o Used by researchers because a part of them is similar to us
Molecules
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Covalent bonds=sharing of electrons
o Nonpolar covalent-equal
o Polar covalent-unequal and partial charges
Noncovalent bonds
o Ionic
o No sharing
Life itself is carbon based
o Carbon forms 4 covalent bonds to 4 different atoms
Hydrogen bonds are vital to life
o Found in water
o Found in DNA
o Stabilize the shape of lysozymes
Hydrophilic interactions
o Molecules that tend to interact with water via hydrogen bond
o Tend to be charged or polar
Hydrophobic interactions
o Uncharged nonpolar molecules that do not interact with water
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Van der waal interactions
o Weaker than H-bonds
o The dipole interactions are very weak and transient
o Distance dependant
pH=-log[H+]
o concentration of hydronium and hydroxide ions
o more hydronium more acidic
o more hydroxide more basic
o 0-7 acidic
o 7-14 basic
o pH of 7.4 is blood…slightly basic
buffers can act as acids or bases
o amino acids
4 elements make up 99% of humans
o H, C, N, O
7 make up .9%
o Na, Mg, K, Ca, P, S, Cl
4 major families of organic compunds
o Sugars
o Fatty acids
o Amino acids
o Nucleotides
Proteins are the most abundant and versatile of the macromolecules
Macromolecules are constructed by polymerization via condensation reactions where a
molecule of water is given off
Break downadd water
Amino Acids
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Amino group
o Acts as a base
Carboxylic acid group
o Acid
Alpha hydrogen off the main carbon
Side chain (R group)
o The only thing that differs in each of the 20 AA
It exists in an L and a D form (enantiomers)
o Our bodies only use the L form
Three groups
o Polar
o Nonpolar
o Electrically charged (ionic)
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Functions
o Protection
o Transport
o Receptors
o Contractile
o And many more
R groups differ in shape, size, and reactivity with water
PKA
o It is the pH where it is half protonated and half deprotonated
o Only the R group matters (amino and carboxy groups always dissociate)
Proteins themselves are made up of clusters of amino acids
Connected by a peptide bond
o Rigid and not very flexible at the bond point
Dehydration synthesis
Hydrolysis (break down)
Repeat of NCC
o The beginning amino and the end carboxy are the only charged part of the backbone
N-teminus and C-terminus
5 levels of protein structure
o Primary
 Amino acid sequence
o Secondary
 Folding alpha helix and beta pleated sheet
 Stabilized by hydrogen bonds
o Tertiary
 Folding as the unit
 All the secondary structures
 Stabilized by:
 Disulfide bonds
 H-bonds
 Covalent bonds
 Van der waals
 All these bonds are between side chains
o Quaternary
 Protein has multiple tertiary structures
o Domain
 Domains are protein substructures that can fold intependently into compact,
stable structures
 Different domains have different functions
 Part of a larger proteins
 Specific function
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 Function can operate independently of the rest of the protein
Protein folding
o Protein folding is often spontaneous
 Doesn’t need any help
o Some proteins need molecular chaperones which give them an environment to fold
o Denature
 Proteins unfold due usually to heat or pH changes
 Disulfide bonds are hardest to break
 Misfolded proteins cause human diseases
 Prions
 β-mercaptoethanol breaks disulfide bonds
 SDS (sodium dodecyl sulfate) totally denatures a protein
Lipid, Membranes, first cells
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Plasma membrane is a lipid bilaryer
o Separates the cell from the outside environment
o Chemical reactions are more efficient
o Selective barrier
o Localized specific functions
Two types of lipids
o Fatty acids
 Hydrocarbon tail
o Isoprene
Lipids have a major hydrocarbon component and are mostly nonpolar and hydrophobic
Steroids
o Ring structure
Phospholipids
o Choline, Phosphate, glycerol, 2 fatty acid tails
 Ester linkage between the glycerol and the fatty acid
o Amphipathatic
 Polar and nonpolar component
o Hydrophilic head
o Hydrophobic tails
Kinks in the fatty acid chains caused by cis double bonds
o Creates more space, allows the membrane to be more fluid
o Found in cold environments
Shorter tails also increase mobility
o Less forces
Temperature increases, fluidity increases
o Vice versa
Very hot conditions? Not many kinks and the chains are longer
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o Vice versa for cold
Long and saturated=less permeable
Short and unsaturated=more permeable
Amphipathatic molecules form micelles or bilayers depending on the shape
o Cone shapemicelles
o Cylinderbilaryer
Permeability
o Small nonpolar molecules get in easy (hydrophobic)
o Small uncharged polar molecules get in
o Large uncharged polar don’t get in
o Any ions cant get in (charged)
Eukaryotic cells add cholesterol to change permeability
o Disrupts van der waal forces
o Reduces membrane fluidity
Fluid mosaic model
o Proteins span across the membrane
Predictions of the fluid mosaic model
o Membranes are made up of 2 layers with hydrophobic chains on the interior and polar
head groups exposed to the exterior
o Mobility of lipids is allowed only lateral and rotational and never a flip flop
o Bilayers are asymmetric in nature
 Differ in lipid concentration
o Membrane proteins and either integral or peripheral
RBC it is no cytoplasm
Can see the bilayer asymmetry through an antibody reaction with both sides
Phospholipase C
o Degrades the phospholipid membrane
Use a detergent to gain access to the inside
Can see the mobility of the phospholipids
o Fusion experiment
o Tagging with florescence
Raise the tempmore mobility
Types of membrane proteins
o Transmembrane and membrane associated
 Transmembrane –all across
 Membrane associated-only one side
 Lipid linked-inside or outside, linked to phospholipids
 Protein attached-one transmembrane protein and another attached protein
If there are 18-21 nonpolar proteins in a row, there is most likely a transmembrane domain
o Usually an alpha helix
Most proteins have a minimum of 3 domains
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Passive vs active transport
o Passive is diffusing without energy down a concentration gradient
o Active requires an input of energy and against a concentration gradient
Linear graph=passive transport
Passive across membranes
o Ions (need a channel)
o Aquaporins (allow water)
o Carrier proteins
Ion channels
o Highly specialized for only allowing a certain ion in
o Concentration
o Or charge
o Gated or passive channels
Saturation rate with transport
o Eventually reach a maximum and it levels off
o Can only diffuse so fast
Inside the Cell
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Prokaryotes
o Not too many organelles (no membranous ones)
o Ribosomes
o Circular DNA
 In a nucleoid region
o Cytoskeleton
 Plays a role in cell division, shape determination, and polarity determination
o Cell wall
 Made of peptidoglycan
 Gram positive
o Thick peptidoglycan wall
 Gram negative
o 2 membranes
 Peptidoglycan and lipopolysaccharide and protein
membrane
 Releases endotoxins
 Harder to defend
o Plasmids
 Able to be shared with other prokaryotes
 Circular
Eukaryotic cell walls
o Fungi, algae, plants
o Stiff cell wall for cellular support
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Semi permeable
 Permits small molecules and small proteins (30-60 kda)
o Plants and algae have a cell wall made of cellulose
o Fungi=chitin cell wall
Eukaryotic cells have membrane bound organelles
o Nucleus
o ER
o Golgi
o Peroxisomes
Secretory cells
o Secrete a product (can be good or bad)
o Make things
 Highly specialized
o Produces material and secrete into a duct or the blood
 Exocrine gland=duct
 Endocrine gland = blood
 Secretion is regulated
 Products are released from secretory granules
Nucleus
o Surrounded by a double membrane nuclear envelope
o Nucleolus=site of RNA/ ribosome assemble
o Fibrous proteins form a lattice
 Nuclear lamina
 Structure and shape of the nucleus
o Function
 Information, storage, and processing
 Contains the cells chromosomes (code)
 Ribosomal RNA synthesis
o Nuclear membrane is an extension of the rough ER
o Things can get through a nuclear pore complex
 Regulates proteins going in and out
 Selective based on size
o Nucleus has a distinct set of proteins that work with it
o Each protein has a targeting sequence within the primary sequence
 Molecular “zip code”
o Nuclear localization signal (NLS)
 17 AA signal that tells a protein it belongs in the nucleus
Rough ER
o Network of membrane bound tubes and sacs studded with ribosomes
o Interior is the lumen
o Rough ER continuous with nuclear envelope
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Hugs nucleus tightly
Function
 Ribosomes in rough ER synthesizes proteins
 Proteins are folded and processed and assembled in the lumen
 Proteins are glycosylated in the ER lumen
 Begin protein modification
 Majority occurs in the golgi
Smooth ER
o Rough ER without ribosomes
o Enzymes within smooth ER produces fatty acids and phospholipids or breaks down
poisonous lipids
o Calcium ion reservoir
Golgi
o Golgi apparatus is formed by a series of flat membranous sacs called cisternae
o Function
 Golgi processes, sorts, and ships proteins synthesized in the rough ER
 Also site of lipid synthesis
 Membranous vesicles carry products to destination (either inside the cell or out)
 Carried in vesicles to the golgi too
o 3 faces
 Cis golgi is closest to the nucleus
 Medial golgi
 Trans golgi-closest to the PM
o The proteins can have destinations
 Resident in the cis golgi
 Continue to go on to inside or outside the cell
o Proteins in the lumen are dumped outside the cell
o Membrane sequence can allow a protein to be transmembrane
o Destinations?
 Lysosomes
 Plasma membrane
 Secretory vesicles
o Add oligosaccharides to proteins that are secreted out of the cell
Mitochondria
o 2 membranes
 Outer
 Inner-cristae
 Folded
o Has its own DNA and ribosomes
 Supports the belief that they used to be cells
o Function
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 ATP production
Peroxisomes
o Globular organelles bound by a single membrane
o Function
 Center of oxidation reactions (contains them)
 Catabolize long chain fatty acids, branched fatty acids, polyamines, and creates
plasmolagens
o Specialized peroxisomes in plants called glyoxysomes
 Packed with enzymes that oxidize fats to be used in energy storage
Lysosomes
o Membrane bound structures containing approximately 40 different digestive enzymes
o Found in animal cells
o Function
 Digestion
 Waste processing
 40 different enzymes break down
 Nucleic acids
 Carbs
 Lipids
 Proteins
o Working pH is 5.0
 Has a pump to maintain a low pH
Endocytosis
o Phagocytosis
 Ingestion of large molecules
o Macropinocytosis
 Membrane grows around
 Fluid uptake
o Clathrin-coated vesicles
 Forms a membrane enclosed vesicle
o Non-coated vesicle
o Caveolae
 Smaller vesicle (50-80 nm)
Lysosomes
o Four processes
 Phagocytosis
 Autophagy
 Endomembrane transport
 Receptor mediated endocytosis
 Early endosome matures to late endosome
o Lysosomal storage diseases
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 Substances accumulate inside of the cell
Endomembrane
o ER, golgi, and ribosomes work together to produce proteins
o Proteins synthesized in the rough ER
o Move to golgi for processing
o Travel to the cell membrane or other destinations
ER signal hypothesis
o Life begins in cytoplasmic ribosomes
o Signal sequence is synthesized by ribosomes
 About 20 AA
o SRP binds to signal, pauses translation, and brings it to the rough ER
o SRP receptor allows translation to continue
o SRP signal is removed
Trans golgi network
o Form a vesicle
o Get a mature lysosome
o Maturation process
Cytoskeleton
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Composed of protein fibers
Gives the cell shape and structural stability
Aids cell movement and transport of materials within the cell
Organizes all of the organelles and other cell structures into a cohesive whole
Disrupt the cytoskeleton?
o Entire intermembrane system is destroyed
3 types of cytoskeletal elements
o Actin
 Along the outside (cortex) of the cell
o Intermediate filaments
o Microtubules
 Organize the organelles
Microtubules
o Made of alpha and beta tubulin dimers
o Have polarity
o Dynamic (can grow and shrink)
o Usually grow from the plus end of the cell
o Function
 Stability
 Movement
 Structural framework for organelles and a track for intracellular transport
o Originate from Microtubule Organizing centers (MTOC)
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 Major one is the centriole in animals
Taxol
 Molecule that stabilizes microtubules to study them
Colchicine
 Depolymerized MT
Motor proteins that use ATPase enzymes to move
 Kinesin
 Towards the + end
 Dynein
 Towards the – end
Form cilia and flagella
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Actin
o Microfilaments
o Grouped in bundles
o Found just inside the cell membrane in the cortex
o Two strands of actin are twisted together for strength
o Play a role in muscle movement
Intermediate filaments
o Provide structural support for the cell
o Not involved in movement
o Connected cell to cell or cell to ECM to help anchor the cell
Cell Adhesion
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Cells are physically connected to the ECM
ECM
o Everything outside of the cell
o Peripheral proteins are usually anchored by actin filaments on the inside of the cell
Integrin (in the PM)
o Attaches to fibronectin in the ECM which binds to collagen
o A way for the cell to anchor
Collagen
o Part of the ECM (main component)
o Most abundant protein in the body
Fibronectin
o Dimer
 Linked by disulfide bonds
o Binds to integrins
Tight junctions
o Forms between PM of adjacent animal cells to form barriers that allow adhering
o Membranes are pinched together and have membrane proteins to form a tight junction
o Usually found in tissues
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o Tight water line
Desmosomes
o Adhering of cell’s intermediate filaments
o If damaged, can lead to blistering diseases
o Proteins in between=cadherins
Gap junctions
o Allow cells to communicate
o Allow small molecules to pass through
o Can be gated or ungated
Selective adhesion
o Cell-cell connections are species and tissue specific
Signal transduction
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Every cell interacts with its environment
o Relay signals from outside in
Receptors
o Found in the plasma membrane for signals that cannot pass through the PM
o Found in the cytosol for signals that can pass through the PM (lipids/steroids)
 Receptor usually becomes a transcription factor and goes into the nucleus
Binding creates a shape change in the receptor, activating it
Target cells are very specific
o Long distance signaling uses the blood
o Short distance signaling is local growth factors
Transmembrane receptors
o Shape change leads to
 Activation of cytosolic protein that trigger the production of intracellular
messenger molecules
 Enzyme linked receptors (Tyrosine kinase) that create a phosphorylation
cascade that activates a series of proteins in cell
G-proteins
o Activated by GTP
o Steps
 Hormone binds to receptor
 Change activates the G protein
 G protein binds GTP
 G protein activates downstream enzymes which signal or generate second
messengers
 cAMP, Ca, cGMP, DAG, IP3
 second messenger cascade amplifies the signal and increases the reaction
o deactivation
 G proteins hydrolyze GTP to GDP
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 2nd messengers degraded or pumped back into storage (Ca ion)
 Cascades are turned off by phosphatases
enzyme linked receptors
o directly phosphorylize kinase by the receptor instead of using a 2nd messenger
o TK form a dimer when activated
o Steps
 Signal binds
 Triggers a receptor cross phosphorylation
 Causes Ras to bind to GTP and receptor through bridge proteins
 Ras triggers a phosphorylation cascade
Phosphorylation by a kinase
Dephosphorylation by a phosphatase
Cell cycle
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Basic detail (see cell bio notes for a complete chapter on cell cycle)
Five steps of mitosis
o Prophase
 Chromosomes condense and the spindle begins to form
o Prometaphase
 Nuclear envelope breaks down
 Kinetochore microtubules connect
o Metaphase
 Chromosomes complete migration to middle of the cell
o Anaphase
 Sister chromatids separate
 Pulled to opposite sides of the cell
o Telophase
 Nuclear envelope reforms and spindle disintegrates
Cell division than begins (cytokinesis)
o Myosin and actin interaction
Mitosis varies somewhat in organisms
o Not always by the 5 steps
Experiments tell us that the chromosome is pulled away from the spindle from the + end to the
pole
o And that the + end disassembles
Cytokinesis
o Once segregated, need to divide the cytoplasm
o Plants
 Cell plate needs to form so that a new cell wall can form
o Animal cells
 Existing membrane is pinched by actin/myosin contratction
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Regulation of the cell cycle
o Signals are given to the cell to start the cycle
o 2 types of proteins in cell cycles
 Ones that are fundamentally required to proceed
 Ones that are only important if something goes wrong and needs to be
corrected
o Mitosis promoting factor (MPF)
 Induces mitosis
 Cyclin/cdk complex
 Cyclin cycles
 Cdk remains constant
Checkpoints
o G1
o S
o G2
o M
o Don’t complete the cell cycle if DNA or chromosomes are damaged
Mitogens
o Proproliferative growth factors
o Influence a cell’s decision to enter S phase
o The RB protein stops s phase entry until it is deactivated by the mitogen
P53
o Guardian of the genome
o Acts as a DNA damage sensor
o Can halt the cell cycle so DNA can be repaired
o Detects mutations
 50% of human cancers have a p53 mutation
Failing to control the cell cycle
o Cancer
o unicellular
 Defect in nutrient sensing allows the cell to be outcompeted for nutrients
 Defect in starvation censing leads cells to grow in the absence of sufficient
nutrients
o Multicellular
 Defect in sensing signals for growth leads to a failure to develop
 Defect in signals to arrest growth would lead to cancer, which is not a single
disease but a collection of different defects
Meiosis
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Sexular reproduction must have some evolutionary advantage
o Puryifying selection
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 Sexual derived progeny are less likely to have deleterious alleles
 They get an evolutionary advantage
o Changing environment hypothesis
 Sexual reproduction gives greater diversity, and greater chance for adaptation
 Asexual reproduction is fine when the environment remains stable
Karyotype-complete set of chromosomes in a cell
Diploid-contains two non-identical copes of each chromosome
One pair of homologous chromosomes
o Same set of genes in the same order
o Contains different alleles
Humans
o 22 autosome pairs
o 1 pair of sex chromosomes
o 46 total
Meiosis halves the number of chromosomes to create haploid cells
Only occurs to create germ cells (egg/sperm)
Tetrad of chromosomes forms
Meiosis I
o Reduces the number of chromosomes
o Daughter cells are haploid but with replicated DNA
Meiosis II
o Stay haploid
o Now have 1 copy of each chromosome
o Not replicated
From one cell, you get 4 haploid gametes
Most important step in meiosis is crossing over that occurs in late prophase I
o Genetic recombination
o Produces new combinations of alleles
Heredity
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Mendel’s model peas
o Self pollination
o Cross pollination
Tests revealed
o Mathematical rules of heredity
o Phenomenon of dominant and recessive traits
The test results contradicted the blending-inheritance hypothesis
o Which was just a 50/50 blend of 2 parents
Control experiments needed for conclusions
o He worked with pure line strains
Polymorphic
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o 2 or more alleles
Particulate inheritance
o Trait maintains its integrity through generations
o Easy to assess
Single phenotype (in this case) is controlled by a single gene
o Dominant
o Recessive
 Refers to the relationship between alleles
 Dominant is expressed in the heterozygote
Wildtype
o Defines a reference allele
o Mutant is a change in function from the wild type
Punnet square is based on Mendel’s observations
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Test cross determines what the unknown allele is
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Uses ratios of the offspring to tell
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Traits segregate independently because the segregation of homologous chromosomes during
anaphase I is random
The genotype in a daughter cell depends on lining up and segregation during anaphase I
o Segregate based on how they line up
Mendel’s genes were all on different chromosomes
o Segregated independently
o If they had been on the same chromosomes they would not have segregated
independently
Sex chromosomes
o Genes present on sex chromosomes are sex linked
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Human y chromosome does not have many genes on it
Males only have one x and one y, so they only display the defective phenotype
o Have no second copy to cover
The closer 2 genes are located on a single chromosome the more linked they are
o Crossing over is rare between them
o Linked genes segregate together EXCEPT when crossing over occurs
Incomplete dominance
o If a genotype is heterozygous, it will appear as a mix of the dominant and recessive
o Close to blending inheritance but the second generation ratios were not correct
Codominance
o Two alleles are phenotypically expressed in equal measures
o Example is blood type AB
Multigenic trait
o Interactions between different genes affect phenotype
Pedigree analysis
o Very useful to helping figure out what disease it is and how it is passed on
o Autosomal-both males and females affected
o Recessive-affected children without affected parents
o Dominant-one parent affected and all children affected
o X linked
 Only sons get the disease
 Daughters are carriers
o Y linked
 Male to male (since cannot get a Y from anywhere else)
DNA and the gene
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Chromosomes are 60% protein (histones) and 40% DNA
Hershey-Chase Experiment showed that DNA is the hereditary material
o Marked phosphorus (DNA) and sulfur (proteins) and saw the presence of tagged
phosphorus in the offspring
Transforming principle (Griffith experiment) confirmed DNA is the genetic material
o Something from the dead virolent can transform nonvirolent into virolent
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DNA structure
o Nucleotide
 3 components
 Sugar (deoxyribose) (5 carbons)
 Phosphate group
 Nitrogen base
o Sugar
 Deoxyribose in DNA
 Ribose in RNA
o Phosphate group is added to the 5’ end
o Sugar base bound to the 1’ end
o Bases
 Pyrimidines-single ring structure
 C, U, T
 Purines-2 ring structures
 Guanine and Adenosine
Condensation reaction is a phosphodiester linkage
o A-T
o G-C
Bond between an O and a phosphate group
2 strands are antiparallel
The DNA ladder twists to form a right handed helix to optimize the interactions between base
pairs
The purines need to bind to the pyrimidines to maintain a constant diameter
RNA is a more versatile molecule because it is single stranded so it can form loops and twist
Replication
o One strand serves as a blueprint for a second strand
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Add to the 3’ end
 Can only go in the 5’ to 3’ direction
Primase
 Creates a RNA primer so DNA polymerase can attach and begin replication
DNA polymerase has a proofread function
Replication is semiconservative
 Not conservative or dispersive
 Found experimentally with Nitrogen markers
 Half and half in first gen, half and half and some completely new in
second gen
Replication begins at replication bubbles
 It expands in both directions
 Prokaryotes have only 1 origin
 Eukaryotes have multiple replication bubbles
 Leading strand is in the 5’ to 3’ direction
 Lagging strand is 3’ to 5’
 Requires many more primers
 Okazaki fragments
o Joined by ligase by covalent bonds
DNA polym I
 Removes the RNA primer
DNA polym II
 Extends the leading stran/okazaki fragment
Problem when the end of the chromosome is reached
 There is a small end that cannot be replicated
 Telomeres
 At the end of the chromosome to prevent the real DNA from being
eaten away
 Telomerase
 A protein that can synthesize the repeats
 Allows chromosomes to replicate without losing valuable genes
 Not found in many cells
 Shortening occurs on the leading strands in addition to the lagging strand
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Changes to DNA
o Replication errors
o Spontaneous damage
o Chemical/radioactive mutagens
o Transposition
o Viral transduction
DNA repair
o Nucleotide excision repair
o Base excision repair
o Mismatch repair
o Double stranded break repair
Excision repair
o Usually thymine dimers
How Genes Work
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Genes code for proteins
DNA to RNA = transcription by a molecule called RNA polymerase
RNA to Protein = translation by ribosomes
3 bases in mRNA codes for a protein
One gene one enzyme hypothesis
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o Proposed that each gene contains the info needed to make an enzyme
Beadle and Tatum used bread mold to test the hypothesis
o They damaged DNA to create mutants that had new nutritional requirements
Srb and Horowitz
o Tested the one gene one enzyme theory using the Arganine pathway
Central Dogma of molecular biology
o DNA is the information storage material
o Sequence of DNA calls for Amino Acids
o The base sequence must first go through RNA before it becomes a protein
mRNA carries info from the DNA to the site of protein synthesis
mutations can occur
Transcription identifies the template DNA strand to limit the choices to the three possible
peptides
Translation can identify which frame to read
A single point mutation in the DNA can have huge effects on the protein
o Ex: sickle cell anemia
Exceptions to the central dogma revolve around functional mRNA molecules that are not
translated and perform other functions in the cell
Some viral genomes also contain reverse transcriptase, which takes RNA and inserts it into the
DNA of the host cell
o Mostly retroviruses such as HIV
20 total AA
o 64 possibilities based on the 3 codons
o Repeats (redundant)
Nirenberg and Leder
o Cracked the codons by creating RNA molecules
o Found that the start codon is AUG
o There are 3 stop codons
 UGA, UAA, UAG
o Many organisms can incorporate a 21st AA
 Selenocyteine
o And some Archaean bacteria have a 22nd
 Pyrrolysine
Code
o Redundant
o Unambiguous (only a few exceptions)
o Nearly universal
o Conservative
Using the Code
o Predict codons and AA sequence encoded by a particular DNA sequence
o Approximate the mRNA and DNA sequence that codes for a particular sequence of AA
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Engineer specific changes to a protein in order to determine the consequences of such a
change
Mutations
o Can be point (one change)
 Missense-one AA replaced
 Silent-no change to the AA
 Nonsense-premature stop codon
 Frameshift-shift the whole frame + or – 1
o Or can be chromosome level
 Polyploidy-increased number of chromosomes
 Aneuploidy-addition or deletion of chromosomes
 Inversion-within the chromosome it breaks off and reattaches somewhere else
 Translocation-moves to a different location
o Mutations can be:
 Beneficial to increase fitness
 Neutral (no effect)
 Or deleterious and decrease the fitness of the organism
Transcription
o DNA to RNA
o Gene expression describes the production of a functional product (RNA or protein)
Different processes regulate gene activity
o RNA
 Synthesis
 Processing
 Degredation
o Protein
 Synthesis
 Modification
 Localization
 Degredation
Transcription is the synthesis of a single stranded RNA complementary to a DNA template strand
H bond between bases
Phosphodiester bonds between sugar and phosphate backbone
RNA is synthesized in an antiparallel complement to the template strand
RNA bonds
o Phosphodiester bond between OH on 3’ carbon and the phosphate group on the next
nucleotide
ORF
o Open reading frame
o Stretch of sequence starting with a start codon and ends with a stop codon
Prokaryotes
o 1 RNA polymerase
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Eukaryote
o 3 types of RNA polymerases
 I-most rRNA genes
 II-protein coding genes, miRNA, plus some of the small RNAs
 III-tRNA and some other types of RNA
Prokaryotes
o Promotor=start signal
 Where RNA polymerase starts the process of transcription
o Attaches to a TATA box
 Found on the nontemplate strand
o A preinitiation complex assembles
 RNA polym II
 CTD
 Other factors (transcription factors)
o Required for transcription to initiate
Prokaryotic Termination
o The presence of the 2’ OH in RNA allows greater flexibility, this allows greater flexibility
to generate hairpin that triggers termination
o A hairpin loop forces a termination that yanks the RNA out of RNA polymerase
Eukaryotic termination
o Processed
o Factors recognize that a sequence is telling them to stop
o Splicing activity needs to occur
 Exons
 Coding (expressed)
 Introns
 Noncoding
 Some DNA does not have complementary RNA (introns)
 Introns are removed by splicing and the exons are spliced together to create
one RNA strand
 Cap and tail are still included
 Spliced by a spliceosome
 Small nuclear ribonucleoprotein (snRNP)
 snRNP ID’s the ends of introns and binds to them
o 3 other things happen to RNA
 5’ end is capped for protection
 Splicing of introns out and exons together
 Polyadenylation –add a poly A tract to the 3’ end
Eukaryotes tend to only code for one protein
o Some exceptions
o Alternate patters of splicing can produce multiple forms of a protein from one transcript
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Enhancers
o Cis acting that bind to trans-acting factors in Eukaryotic cells
Prokaryotes translated right as its being transcribed
Southern blots=DNA
Northern blots=RNA
Translational
o Synthesis of proteins from an RNA template
o Take the RNA molecule outside of the nucleus to the ribosomes in the cytoplasm
o Ribosome needs to recognize mRNA as a substrate and reading it
tRNA
o 3’ end is the amino acid acceptor arm and has an internal anticodon base pairs with
codons in the mRNA
o Binds to and brings a tRNA to the ribosome
o 3 loops for a tRNA
 2nd loop contains the anticodon
o Net result-AA is selected by its codon
 Creates a high energy bond between 3’ and AA
 tRNA becomes charged when bound to an AA
o some tRNA can wobble
 means that there can be a mismatch in the 3rd position of codon-anticodon pair
 explains why there are fewer tRNA than codons
Inosine
o 5th Nucleic Acid
o Modified derivative of Guanosine
Ribosomes
o Eukaryotic
 49 ribosomal proteins, 3 different rRNA molecules make up the large (60s)
ribosomal subunits
 33 ribosomal proteins, 1 different rRNA creates the small (40s) subunit
o Creation of ribosomal subunits takes place in the nucleolus
o Assemble into a full ribosome in the cytoplasm when bound to an mRNA
rRNA
o don’t code for proteins
o transcribes from genes
o recognize and bind RNAs and proteins
o catalyze the peptide bond between AA
o 80% of RNA is ribosomal
Peptide bond formation lengthens AA chain in the 5’ to 3’ direction
E, P, and A sites in the ribosome
o A is for aminoacyl site where the tRNA enters
o P is for the peptide site where the bond forms
o E is for the exit site
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Peptide transferase reaction
o Accomplished by the transfer of AA from tRNA in P site to the A site
Eukaryotes
o The 5’ cap binds to the 3’ tail and creates a loop, this allows the binding of the small
ribosomal subunit to the 5’ end
Shine dalgarno
o Ribosome binding site in bacterial RNA (mRNA)
o Specifically tells what reading frame (since there are multiple ones
Termination
o Release factor binds to stop codon
 No charged tRNA
 Special release factor
o Will cause a release of polypeptide
o Large subunit is first to leave
In bacteria, transcription and translation are tightly coupled allowing for a mode of transcription
regulation based on availability of certain tRNA
Can transcribe and translate at the same time
Eukaryotes separate transcription and translation in time and space
Molecular chaperones
o Help proteins find their correctly folded shape
o Bind to hydrophobic surfaces
o HSP70 (heat shock protein)
 Helps the protein refold after it denatures
Gene Expression Prokaryotes
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Gene expression can be regulated at any stage from mRNA synthesis to protein activity
Types of regulation
o Transcriptional
 Saves the cell the most energy
 Don’t transcribe all the genes
 Only the ones that need to be used are transcribed
o Translational control
 Least common
 Used to control levels of some ribosomal proteins
 Controls whether or not ribosomes will translate mRNA
 Charge mRNA life span
 Change translation rate
o Post translational
 Speed of response is of the greatest importance
 Energy is expensive but creates a fast response
 Proteins are activated/inhibited by chemical modifications
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Proteins get made in the cell but the activation signal usually comes from
outside
There are many control points of gene activity
Transcription controlled
o RNA abundance is altered
o Protein abundance is altered
o No change in other aspects
Translational
o RNA abundance is not changed
o Protein abundance is altered (does not translate)
o Other aspects unchanged
Post translational
o RNA no change (already a protein)
o Protein abundance changes
If RNA levels change…
o Can figure out if it is reduced transcription or increased degredation
o To differentiate, we block transcription from taking place
 Learn about how fast RNA is being degraded
Transcriptional regulation: Protein-DNA interactions
o A transcription factor is a DNA binding protein that influenced the level of transcription
of a gene, either positively or negatively
o They bind to promotors
o Specific minor/major grooves that recognize specific binding sites on DNA
Consequences of protein binding to DNA
o Hinder RNA polymerase from recognizing the promotor region
 If a repressor is bound to an operator region no transcription occurs
o An activator may bind near RNA polymerase promotor site
 This draws the RNA polymerase to the promotor region
Repressors or activators are referred to as trans acting factors
Site where they bind is a cis acting element
CIs mutations
o 2 mutations on the same DNA molecule
Trans mutation
o 2 mutations on different DNA molecules
Use cis/trans test to determine whether or not mutations are present on the same gene or not
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Cis dominant mutations only affect the function of the DNA molecule on which they are located
o Haploid-automatic
o Diploid-have another to cover, can be recessive
o On the same strand
 Phenotype is wild type and the function is limited by the genes acitivity that is
mutated
Trans
o On different strands
o Elevates phenotype
o Mutation on upper chromosome appears to be dominant
Constitutive mutations
o Always on
o Express the gene even when the promotor is not being targeted
Catabolite repression
o Form of feedback inhibition, the product of a pathway inhibits upstream components of
the pathway
Back to translational control
o N-end rule
 The amino end of the AA determines the half life of the protein
Synthsis
o mRNA binds to small subunit via the ribosome binding site
o F-Met tRNA binds (initiator codon)
o Large subunit binds
Secondary structure of the mRNA can hide the Shine Dalgarno (SD) sequence so that the small
ribosomal subunit does not bind, and translation is not initiated
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o Basically the RNA itself is regulated instead of the ribosomes
The SD sequence also can form a stem loop to block access
o Called riboswitches
 Thermosensative
 Could respond to proteins too
Feedback regulation tells the cell to stop making a certain product if there is enough of it
o Saves the cell energy
Also a regulation to the presence of ribosomal subunits
o Need an equal amount of the small and large subunits
Post translational
o Protein phosphorylation
o phospho-relay (cascade)
o Protein methylation
Detect post translational modifications
o Western blots can detect a mobility shift
o If a mobility shift is observed one can use an enzyme to remove the modification and
see if the mobility ceases
o Mutating the predicted site of modification should alter the mobility
o Tag the proteins with antibodies
o Analyze using mass spec
Gene expression Eukaryotes
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Variety of mechanisms to control gene activity
o Chromatin (unique)
 Protein/DNA complex
o Nucleus
 Chromatin remodeling
 Euchromatin-active
 Heterochromatin-inactive
 Transcription – pre mRNA
 RNA processing
 Cap and poly-A tail
o Cytoplasm
 mRNA stability
 translation
 post translational modifications
every step from heterochromatin to active protein can be modified
chromatin
o histone protein and DNA
o 8 subunits of histones with DNA wrapped = nucleosome
o Organized DNA
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o Nucleases can take DNA off for translation
Chromosome is interspersed with hetero/eu chromatin
Condensed  uncondensed
o HAT (Histone acetyl transferase) adds acetyl groups to decondense chromatin
Uncondensed  condensed
o HDACs (Histone deacetylases) remove acetyl groups to condense chromatin
Enhancers in a gene are binding sites for transcriptional activators that are not near the
promoter
o Can be found upstream or downstream
Core promoter binds to general transcription factors
Promotor proximal elements are binding sites for transcriptional activator or repressors that are
near the core promotor
If an enhancer is translocated to a different place, it can enhance a different gene
Transcriptional control allows cells to respond to environmental changes
Epigenetic inheritance
o Inherit the same patterns of histone modifications
o Have same hetero/euchromatin
X chromosome inactivation in women creates 1 barr body
Transcription initiation depends on an initiation complex
o The TF binds to DNA
o Chromatin loosens
o TF bind to enhancers and promoter proximal elements
o Basal transcriptional complex begins transcription
DNA is dissociated from the nucleosomes by RNA polymerase
Post translational control
o Once an mRNA is made, a series of events occur if the final product is going to affect the
cell
o 4 major control points
 Alternative splicing of exons
 Takes place in the nucleus
 Benefit is that different forms lead to different domains that can lead to
different functions
 Alter the rate of translation initiation
 Alter mRNA stability
 Post translational modifications to alter protein abundance, localization, or
interactions
RNA stability and RNA interference
o In the cytoplasm, there are mechanisms to control gene expression
o Regulate the amount of DNA translated
 Block access to mRNA by ribosomes
 Alter stability of RNA
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Highly variable
o Some degrade rapidly, others are very stable
o Life span of mRNA is controlled by RNA interference
 Specific mRNAs are targeted by microRNA (miRNA)
 Binds to parts of the RISC protein complex
 Degrade the mRNA or prevent translation
o 20-23% of all animal and plant genes are regulated by miRNA
mi vs si RNA
o miRNA
 dsRNA from an endogenous gene
 no perfect base pairing
 mRNA level is unchanged
 there is a reduction in protein
o siRNA
 dsRNA from a foreign source
 perfect base pairing
 leads to RNA being broken down
 mRNA levels reduced
 protein levels reduced
translation is controlled
o miRNA blocks translation
o mechanisms can control the time and rate of translation
o translation can be slowed or stopped by phosphorylation of ribosomal proteins
post translational
o speed of response is fast
o cells can respond to new conditions rapidly by activating or deactivating proteins
o types
 chaperone protein
 enzymes can modify proteins by adding groups
 Proteins can be activated/deactivated
 Targeted protein destruction