Class 1
Cells are the smallest unit of life
all living things are made out of cells the basic units of biological structure
Cell is the smallest biological system
Parts are arranged and organized
Life is an emergent property of cells
Living systems can: reproduce, growth/development, metabolism homeostasis,respond to stimuli, and they
evolve (must have all these properties to be considered alive)
(Where do all living organism come from? Cell theory
• All living cells are formed by the growth and division of existing cells
• all share a common ancestor inherit features from common ancestor (unity)
• some common features but also unique
Eukaryotic vs prokaryotic
Pro-bacteria and archaea
• Pro are much smaller
• all eukaryotic cells have a nucleus
Homology - cells share common characteristic because of common ancestor
Diversity- out of common background a tremendous array of different cell types has evolved
Eukaryote- true nucleus, unique in having organelles
All cells share: DNA o plasma membrane and ribosomes
• universal genetic code I many biochemical pathways and ribosome for protein translation
Homology - sequences are highly "conserved"
• supports the idea of a common ancestor, passed down a common genetic code to all organisms
• mutations happened but never survived
• protein must be essential to life and play important role, almost no changes in them nano acid
sequence have evolved
Evolution of eukaryotic cell
Change over time, inherited change to a population over multiple generations
Macro-new species form
Evolution of eukaryotic cells
Do not know about abiogenesis the evolution
from life from nonlife
Theory of endosymbiosis
Are bacteria and one time bactino pumped ATP and
instead of it being kicked out it stayed and evolved
Class 2
Cell theory-all living cells are formed by the growth and division of existing cells
Leads directly to homology and diversity
01/25
Cellular evolution: a. new player in the story
Phylogeny shows recent common ancestors
Eukaryotes and archaea are more clearly related
archae is the missing link between prokaryotes and eukaryotes
How does a cell evolve and differentiate into a particular cell
· cells must be able to communicate with each other
· respond to changes in the local environment
· function to support life and maintain homeostasis at the
level of the organism
↳ one fertilized egg gives rise to many organisms
.
Specialization
· determined cells -early on cells will figure out what they will be (get a hint at molecular level of then
express it through proteins
cannot differentiate and divide
genomic equivalence -all cells of an organism have same exact genome
· different cells turn on different subsets of the gene
· proteins make the cell what it is (functions)
-all cells turn on the housekeeping genes (exGAP)
-each cell then also turn on its own special subset of gene
· molecular and cellular level must be coordinated w/what is happening at the level of the organism in
its environment to
maintain the organism's homeostasis
-single cell type w/single mutation could lead to a horrible outcome
Cancer Cells
· Change in gene expression
-lose ability to program cell death
-unregulated cell movement
-unregulated cell division
-loss of response to signals
-adapt to new environments
-genetically unstable: increased mutation rate
-soak up all glucose
-Can alter environment
· day 0 to 200 wild type remained tumor free
· day 0 to 110 some of the mice began to get tumors
· Ras day 20 tumors start to appear, half of them
-normal roles of myc and ras is to prevent tumors
* tumor surpressor genes
By the end all mice get tumors if you knock out both genes
Cancer is a result of multiple mutations, they do accumulate and you need 13 different mutations in a
singe
Cancer cells have a selective advantage
The chemistry of life
• All organisms are carbon based I carbon can create 4 covalent bonds)
• most biological processes happen in water
• super complex (water is an excellent solvent and water has a
temperature stabilizing capacity
01/29
Class 3
Covalent bonds are the strongest and most stable (leads to stable interactions)
• many of interactions are non covalent need this for structure of proteins, in order to be flexible
changing of any molecules
• covalent bond-completely sharing e - (can have complete and be nonpolar) (polar hydrogen and
oxygen e- spends more time orbiting one molecule, difference in charge)
Ionic attractions - one has one extra e - positive and negative charge (a salt)
Van Der walls - all atoms
Hydrogen bonds- any time hydrogen in a polar covalent bond next to another polar covalent bond
(between water molecule and more)
All 3 bonds are hydrophilic (easy to dissolve in water)
They exclude interacting with water
Water
At a very low frequency one H+ from one H20 molecule can break off and then immediately join with
another H2O molecule (one in 10 million will do this)
Convert to PH - log [10] 7
Acid-an molecule that has a tendency to dissociate hydrogen then pure water molecule
=
Amino acid- has at least one carboxul group
Bases-have a tendency to pick up H+ atoms, like amino groups
Amino acid- have carboxyl
Strong acid- love giving out hydrogen
H
C
With nc1 all of the HT dissociates from the cl- in the solutionHCl
Weak acid can go either way (some equilibrium)
Cells use weak acid/bases to regulate PH
4.75 = PKA acetic acid
PH too high? The ha can let go of some H+
Ha-weak acid
A "conjugate base" pH to o law? The A can sop up some of ht
. Buffer-the combination of any weak acid (ha) with its conjugate base (A)
Macromolecules
• same chemical reaction to link two things together
• add water to split bond (hydrolysis) generates energy
•
•
=
+
+
+
-
Class 4
Carbohydrates
Monosaccharides-building block
• plants grab co2 out of air
• simpal sugars are used for energy
February I
Glucose
C
Carbon here>
d
C
7
Ei
# I Carbon
LaDglucose
covalent bond betw disaccharide
facing
-Since OH faces down a 1. 4 glycosidic bond(pink)
-level of detail is linked to function
-Carbs serve as energy storage (polysaccharides), a link (alpha)
-also serve for structure B link (beta)
-enzymes read this (cell-cell signal) communication
C
must be
G
down
-oligosaccharides covalently linked to proteins or lipids itself (always facing outside)critical for cell-cell
communication
amino
· carboxyl
· surface proteins are required for binding
· hydrogen
LG 5. a Proteins
Polymers of amino acids
* critical for
• at PH 7 most amino groups are ionized
• side chains: uncharged polar, nonpolar, Structure of protein
• carboxyl behaves like acid
• C bonded to C is nonpolar
• r groups have non polar bonds
• cystine and cystine will find each other and bond covalently (this bond is super strong)
When an amino acid is alone it con dissociate (not in a polypeptide chain)
When peptide bond are formed there is no opportunity to gain or lose an H +
R groups are never involved in the formation of the peptide bond
• side groups can be charged or not
• all weak acids and bases are at equilibrium
• acidic 10W PH (it varies tremendously )
&
pH
=
pKa
log[A]/[HA] "do not memorize
+
We any call it a proper when it has the structure to perform its function ton
Buffer-any weak acid and its conjugate base
no
logka
-
:
log
log[Hi]
PH
=
=
I
-logka
pla
+
+
equilibrium
for pKA
log
logA
A
only
weak
acids
PH : pkA of that molecule
to be a good buffer
Class Meeting 5 February 5
· primary level-sequence of amino acids from N terminus to Cterminus
-all other levels of protein structure are dependent on primary structure Disrupts non covalent bonds
Secondary structure
tertiary structure
* soon as it is in urea it is denatured
Different r groups behave differently
2nd structure-small numbers of amino acids folding
3rd structure- overall 3D shape
Hydrogen bonds are needed to form a helix and beta pleaded sheet
Single stretch of amino acid (a helix)
Multiple stretch of amino acid lining up next to one another
Flexibility
&
B plated
Sheet
* A change in the primary structure of a protein can affect the secondary and even tertiary structure
Domain-any region of a fully folded polypeptide that has a function
Quaternary structure-two or more separate polypeptide (called) subunits interact to form the final protein
Subunits - two separately synthesized polypeptides that now get together
Many things have to happen
All proteins have to fold into the correct shape
Fold based on primary structure (r groups)
Proten synthesis does not happen all at once
Chaperone proten
• critical in helping proteins fold and guiding proteins to correct partner
• any protein not folded correctly will be degraded
Chaperone
HSP 60
HSP 70
HSP 70
• first one to sweep in (during translaton)
• jump on to polypeptide and covers an area of
hydrophobic regions
If it is still misfolded 1 HISP 60 steps in after translation
Repeat folding till we get correct structure
Aggregates are toxic resistant to protease
Very important to correctly fold
Who gets destroyed
• Misfolded proteins
• Proteins that are taking too long
• protein that have finished their job
Polypeptide called ubiquitm to determine
Confusing HW Question
3 = 8 95
+
.
Another Example
3
log A
=
10 53
+
.
109A
HA
HA
-
5 95
=
.
logA
-
7
.
53
HA
A
=
1
.
1x10-3
=
HA
more
=
logA
1 1
.
+
10
-
3
HA than A
no
charge
-
I
charge
COOH* COO
Carboxyl
=
O
amino = I
-
+
H
+
+
charge
Class 6
February 8
· proteins can escape both systems, evolution tries to prevent
· resistant to
proteases
-only particular class of proteins form aggregates
- fold into a very stable structure "amyloid""Cross beta filament"
One misfolded protein can cause other normally folded proteins to misfold
• mad cow disease is infectious
• pnon-any protein that can misfold into amyloid structures and be contagious (very rare)
Enzymes
• speeding up chemical reactions
Potential-stored energy ready to do work stored in
glycosylic bonds, covalent bonds when energy is released as
kinetic energy it allows for the movement of molecules
Most enzymes are proteins
Selective binding interact specifically with
particular substrates
Reactions go from A to B because it must release
energy. Reaction must go downhill
Y axis amount of energy (free energy) G
Y axis time
Must be exergonic substrates must have more stored energy
&
&G
Products Reactants
2
7
5
G
AG
&
12
10
activation
-
=
energy
-
=
=
8
-
6
Enzymes cannot change free energy in products or reactants
Active energy-energy put into the system to allow the
reaction to occur from r to p
Enzyme lowers activation energy and decreases time frame
for a reaction
4
E
2
O
Speed and specificity - interaction between enzyme and substrate
When substrate and enzyme interact we get an induced fit
Allows us to compare
February12
Class meeting 7 (Last Class Material for Exam 1)
• enzymes are all about speed and specificity
Enzyme kinetic
• lowers activation energy
• max - the highest rate in presence of "saturating"
• km - concentration of substrate present to set enzyme to 1/2 vmax
Great way to visualize what is gong on
Enzyme catalyzed reaction:
• really fast at lower concentrations of S
• does reach saturation at higher
concentrations of S
Uncatalyzed reaction
• 10W speed with physiological concentrations of S
• - does not reach saturation at a higher
concentrator of S
Y
int =
umax
Xint
=
-
kM
Enzymes are typically proteins that can be regulated
• turn on enzyme gene
• regulate activity
• cells con manipulate how much substrate is available ISI
2. Making molecules that stimulate or inhibits E
Competitive inhibitor binds to active site
• shut the enzymes down (poison)
• will alter the shape of the graphs in predictable ways
• no product formation
Non competitive inhibitor
• inhibitor and substrate bind to different sites
Allosteric regulation
• binding of a molecule to a site on the protein
that is not the protein’s substrate binding site
• causes a conformational change that can
change the protein's function in some way
(better or worse)
• km does not change
The cell will adjust the pH to ensure A and ha
forms
Exam 1: learning goals
Learning goal la: cells are the smallest unit of life and share fundamental features: all cells are thought to
have evolved from a common ancestor
Seven characteristics to define a cell: reproduce, grow/develop, metabolism, homeostasis, respond to
stimuli, and evolve
Emergent property: properties that become apparent and result from various interacting within a system
but are properties that do not belong to the individual component themselves
Cell theory - all living cells are formed by the growth and division of existing cells, all share a common
ancestor and inherit features from common ancestor (unity) some are common others are unique
Eukaryotes and archaea are more clearly related and archaea is the missing link between
prokaryotes and eukaryotes
Eukaryote - true nucleus, unique in having organelles
Prokaryote-much smaller
February 19
Class meeting 9 exam 2 material
Lipids: a structurally diverse group of molecules, not made linking monomers together via dehydration
synthesis
Not soluble in aqueous solution because overall they are hydrophobic
Found in membranes
saturated
H
HHH
↓
3 categories
I
I
I
I Fats/storage
c
c
C
C
H
2 Membrane
↑
3 Sterols
unsaturated
Fatty acids: the building blocks for storage and membrane lipids
Amphipathic:charged on one side but not the other
Length of hydrocarbon tail and presence or absence
• the longer the tail the more tightly fatty acids interact with one another
• the higher the number of saturated c-c bonds the more tightly fatty acids interact with one
another
Individual fatty acid
Membrane lipids: in particular phospholipid built by modifying triglycerides
• removed one fatty acid
• added a phosphate
• added another highly polar group to phosphate
Glycolipid
•
=
-
-
Sterols
Four members ring (all hydrogen bonds)
Cholesterol is found in the membrane
Lipids make hydrophobic barriers
Membranes: creates compartment of cells
Hydrophobic barrier
1. Barriers
Proteins make it semi permeable
2. Sites for specialized functions
3. Mediate communication with cells and environment
Fluid mosaic model
• fluid because hydrophobic interactions between hydrocarbon tails are very weak
• many different proteins are stuck into the membrane creating a mosaic of proteins
throughout the membrane
Lateral shifts: no problem
Transverse : Less likely
Tm = temperature above which a
particular lipid will liquify and below
which it will literally solidity
The higher the saturation the higher the transition
temperature
Cholesterol: stabilizes fluidity over a range of
temperatures
High-more rigid cholesterol itself is rigid keeps membrane
lipids from flying around
Low-more fluid prevent tight packing of hydrocarbon tails
Class meeting 10
February 26
Learning goal 8
Cells regulate the fluidity by putting different combinations of phospholipids
Cells can make plasma membrane asymmetric in order to maintain homeostasis
Where do cells get all of the lipid components
• food transported through the blood and transported to cytoplasm
• components all gather in cytoplasm
Scramblase
Where are membrane made?
-Outer to inner, non selectively
• smooth Er, site of membrane synthesis
• new membrane lipids are made by enzymes facing the cytoplasmic side of the SER membrane
• then those enzymes insert the new lipids into cytoplasmic leaf of the SER membrane
• then some of that membrane is distributed to the other organelles and even the plasma membrane
Scramblases vs Flippbases
· flip a phospholipid
Flippbase
Cholesterol can tip back and
• selectively
forth pretty evenly distributed
Inner membrane is more fluid than outer membrane
Membrane proteins
• transporter and channels
• anchors
• receptor
• enzyme
• integral membrane (transmembrane proteins)
• peripheral (polar heads of lipids/ surface)
• covalently linked to a lipid
Integral membrane protein
• a transmembrane domain = membrane spanning
domain
• alpha helix are more common
Multi pass protein also have alpha helixes and beta
pleated sheet
Learning goal 8B
• Unregulated simple diffusion
1. F and A
2. Small non polar solutes (diffusion) get
through on their own
3.Simple and facilitated
4. Active transport ad facilitated diffusion
High to low is with the gradient
Channels are selective (only potassium ions)
Channels and transporters facilitated
diffusion high to low
The pump is from low to high so energy is
required ( active transport)
Simple - does not involve protein
Class meeting 11
· GLUT family of proteins that allow facilitated transport
-When gradient is gone over time We go back to where it started
-must go down the gradient
against the gradient-Use energy,
· energy source depends On PUMP
· high energy bonds
· gradients also store energy
- pumps -transporter
proteins that move solutes
against their gradient
NatIK "pump-bind to and hydrolyze ATP, pumps sodium
and potassium against their gradients (creates and maintains 2
gradients)ONE PUMP always working
· cycle is constantly happening
· results in maintaining 2 concentration
gradients Na+ (high outside)and K+ (high inside)
· creates a maintains charge gradient cytoplasm is slightly more negative
membrane potential-difference in charge across a membrane
Gradient driven pump:
Nat/glucose-glucose transporter, will have
sodium potassium pump
Apical
Glucose transport
Basal-faces blood
Cancer cells take huge amounts of glucose (they are relying on glycolysis)
Glucose homeostasis
Warburg effect - in cancer cells they will switch from aerobic
respiration to using glucose
Channels
Hole for the membrane, passageway down its gradient
Ions
K + channel
Selective designed perfectly for particular work by facilitated
diffusion and are gated
Ligand gated
Mechanically gated channels
Class meeting 12
flow of ions along the axon is the electrical impulse
Amount of charge inside vs outside of cell resting membrane potential is negative
Small flow of Na + ions will do nothing but large amounts will wake up channels
Now a whole lot more Na t flows in through channels
*all or none response
The flow of Na through the cytoplasm propagates the electrical signaI down the axon
How do we get back to normal?
• positive charges plummet
• voltage gated NT channels are pre programmed to slam shut,
now in inactive conformation
• voltage gated potassium channels want to go down its
gradient in order to get repolarization
• the same stimulus that opened Na channel also open K
channels but they are just a bit slower. K+ flows out of cell
End of neuron
Calcium in the cytoplasm is always low
• focus on the terminus
• convert electrical signal to biochemical signal (neurotransmitters) proteins made by the cell
Increase in calcium causes membrane fusion
Acetylcholine receptor is a Na + channel
How do we turn off this signal?
• cat 2 channels slam shut after 1 m-sec
• it is destroyed by acetylcholinesterase
Learning goal 9a bioenergetics
Catabolism is overall exergonic
Anabolism is overall
metabolism is the sum total of all the
chemical reactions in an organism
Connect reactions
Catalyzed and coupled with the help of
enzymes
ATP links these reactions
The breaking ad making of ATP is coupled to
specific reactions in our pathway that
phosphate is temporally linked to a reactant or
product
• coupled means one enzyme carries out
both reactions simultaneously
Step 6 in glycolysis
• very energy producing
• redox reaction oxidation and reduction
• h- hydride oxidation removing, reduction is adding
• hydrogen needs to bind
Learning goal 9b
• need abunch of enzymes to convert one
sugar into two pyruvates
Step l
Phosphorylate glucose
Made a molecule with more energy stored in it
Class meeting 13
Learning goal 9b
Glycolysis - 10 enzymatic reactions in the cytoplasm of the cells
Step I hexokinase will add a phosphate by using an ATP
Any enzyme that adds a phosphate to a substrate is called kinase
It is negatively regulated by glucose 6-b negative feedback regulation)
We would wanna slow down the enzyme if there is too much
Step 2
Enzyme rearranges some atoms
6 membered ring to a I membered ring
Step 3
Add phosphate
Using up second ATP
Phosphofructokinase negative feedback inhibition
ATP can bind to either catalytic domain or allosteric site
Lower km less substrate needed to do its job
Step 4 and 5
Add 2,3 carbon company's
2 high energy compounds DHAP AND GAP
March 7
ATP
fructo
preferential binding to active
site
to active site
So me will bind
bind to allo
So it will also
it down
Steric site and slow
Step 6
Gap denyarogenase
Oxidation reaction, the only redox reaction in glycolysis
Generating NADH No ATP is being used but still building energy
Steps 6-10 happen twice
Step 7
Phosphate is removed by enzyme
Substrate level phosphorylation - when an enzyme takes a phosphate from a substrate and attaches it to
an ADP
Even on ATP ' must try to make a surplus
Step 8 and 9
Tinkering to optimize the removal of the P
Pep is our 2nd compound (along with gap) that has a high energy P bond
Step 10
Second substrate level phosphorylation
We are done with glycolysis
•
The product of an earlier reaction in glycolysis builds up and stimulates an enzymes activity
Learning goal 9c
If oxygen is available will be completely oxidized and continue
If no oxyge- cell must try to keep glycolysis going stay in cytoplasm and recycle nad
Begins with pyruvate brought into the mitochondria and will get oxidized
Oxygen helps because we can contrue to what happens in the mitochondria
Aerobic respiration
The citric acid cycle
more NADH
Fed into cycle by being attached to y carbon compound
Energy from all of the redox reactions is used to create a gradient which is used to make ATP via oxidative
phosphorylation
The election transport chain
Class meeting 14
How do all cells construct themselves? How are different cells made different?
Learning goal 11
Central dogma: information flow from DNA to protein ( focus on nuclei acids)
• DNA and RNA are made by monomers ( phosphodiester bonds)
• single stranded DNA
• DNA vs RNA
• thymine in DNA and uracil in RNA
Rules for any Double strand
• constant diameter with 2 ring purines with I ring pyrimidines
• hydrogen bonds always matches up
• anti parallel
March 18
In eukaryotes the DNA is packaged
• chromatin - eukaryotic DNA and its associated proteins
Chromatin structure
• 5 different histone proteins
DNA and history proves associate so tightly because of ionic bonds
Small genes in the regions are unwound
Chromatin remodeling
• enzymes that chemically modify histone proteins
1. Acetylation of histones makes history protons let go, bind less
2. Methylation
3. Phosphorylation For positive regulation the goal is to move histones out of the way
• regular transcription factor binds to and brings in
Learning Goal IIC Decoding Info in DNA
• repetitive DNA
Transcription initiation in eukaryotes
• regulatory transcription factors bind to a sequence explanation for differential gene expression)
• decide where genes are on and what time inside which alls and at what level
Promoter-same core sequence, upstream of where +1 is
Enhancer-different sequence, position independent because DNA can loop
Regulatory transcription factor make a promoter available
• rtf proteins have a binding domain
• protein protein interaction domain
Last class for exam 2
March 21,2024
Class meeting 15
Learning goal 11D
• Cis regulatory element - any nucleotide sequence (promoter and enhancers)
• trans acting factors- any protein ( coming from some where else)
• must have promoter for any transcription to occur must be in front of the gene, enhancers do not
matter where they are they are still capable of binding
• low level transcription for silencers
Transcription initiation in eukaryotes
• the rtfs have done their jobs, chromatin is remolded mediator is bought in
Transcription - elongation and termination
• elongation : RNA polymerase II continue to bring
in complementary NTP'S and covalently link them
to the 3'0H of existing NTP
Learning goal 11E
• during transcription as RNA is being made the RNA is being processed /altered (RNA processing)
• the pre mRNA is the direct product of transcription it contains allot the sequences in the DNA
template
• have to get RNA out to cytoplasm for transcription to occur
• RNA processing happens simultaneously
Additional stops reduced in eukaryotes
• RNA processing
• RNA export nuclear transport)
• mRNA degradation
• translational a post translational control
Splicisome
• made out of 6 complexes that have their own small RNA and protein
• RNAs have catalytic activity
•
Bio 211 class meeting 16
March 25
Transcription in eukaryotes - during transcription pre mRNA is also being processed to convert into final
messenger RNA that is used for translation
Information in one strand of DNA is copied into a single stranded RNA by transcription
How can splicing contribute to regulating gene expression?
• provides eukaryotic cells with another way to regulate gene expression
• turning on or off particular groups of genes that will influence what final proteins are present in that
cell type
• every step in gene expression can be regulated by the cell, cell has many ways of influencing what
kind of protein is sitting in the cell which defines what cell will do for a living
• regulating splicing- another example how we can lead to differential gene expression "alternative
splicing" different groups of proteins present in different cells
regulating splicing - cells can choose which exons to link together (different in different cell types)
•
• In prokaryotes Messenger RNA was a direct copy of one of the sequences in DNA. No RNA
processing in prokaryotes. RNA sequence was the same sequence as the sequence found in the non
template strand of the gene
• in eukaryotes they would find DNA sequence and just messenger RNA that contains all exon
sequences all lined up next to each other to make that mRNA
• ^ also saw different combinations of exon and intron sequences, one pre mRNA ( direct copy) saw
many different possible ways of combining exon and intron sequences (alternate splicing)
• results of alternate splicing: different mRNA when translated will translate different proteins,
influences what proteins are present in the final cell (differential gene expression)
• certain part of proteins are identical but others are completely different
• splicing looks for 5' splice junction and link to 3’ splice junction (default if there is no regulation but
there are other proteins inside the cell that may say "Skip that first intron I will cover it up" 5' splice
junction will not be visible and will go to next one
• other different proteins will come in and regulate what the splice machine can see in a different cell
type. Regulatory splicing factors are like regulatory transcription factors they are not going to just
produce the standard they are gong to regulate what happens in a given cell type
• results in one gene ( which was transcribed into single pre mRNA) can be spliced in different ways
• in many different proteins can be produced from the same single original gene
• eukaryotic proteome (proteins in a genome, DNA in an organism) » prokaryotic proteome because in
prokaryotes gene can only make one protein which results in greater complexity in eukaryotic cell
types because they can make hundreds of different protein
Rules for splicing
• exons must be added in order (RNA comes out of polymerase from 5' to 3' so all it can-do is add
them in order cannot skip ahead and grab exon all the way at the end and put in front) must be 1,4,6
• 5’ cap must be present that will protect that end which is found in the exon #I must be included in
every transcript cannot start with anyone but exon 1
• the last exon must contain the hexa nucleotide (aauaaa) or it will never stop making mRNA can
sometimes have more than one
Glycolysis
• intermediates glucose and pyruvate
• enzymes (3 of them are regulation) hexokinase , phosphofructokinase, and pyruvate kinase
• gap dehydrograre (step 6 redox reaction reduce NAD)
• 1 and 3 use ATP
• 7 and 10 produce ATP
Steps in gene expression
• RNA processing (happens in nucleus during transcription) we also have to get RNA out of nucleus
The nucleus
• two membrane Inbounded (one lipid bilayer in inner nuclear
Membrane and another one lipid bilayer in outer nuclear
Membrane) together they are called the nuclear envelope
boundary around nucleus)
• prokaryote with just dna …little bits of plasma membrane
pinched Off the vesicles and stuck to outside of chromatin by all
fusing together we will get two membranes (reason why nucleus
has double membrane) nucleus is NOT a result of endosymbiosis
• outer membrane of nuclear envelope is continuous with Er
Inside
• Cross section-transmission em (tem)
• chromatin is the main residence of the nucleus (DNA associated protein) in a regular cell (not dividing)
wrapped up to level of solenoids all nucleosomes are taken and wrapped up again and looped into
larger domains “loop domains”strong staining is chromatin (dark black and grey) inside nucleoplasm,
some of chromatin is squished up on the outside and it stains most darkly
• heterochromatin - the most densely highly packaged chromatin (repeated DNA packaged much more
tightly since it is not used for transcription) darker and denser
• euchromatin - the rest of the chromatin (true chromatin which is used for transcription, can be
unwound in a given cell , less dense less staining)
• would not be able to see nuclear envelope without dark staining squished up on the side
• nuclear matrix are all about structure, protein complex networks that provide structure and support
to nucleus holds it in its 3 dimensional shape (skeleton of the nucleus)
• nuclear lamina complex of proteins, its job is more specific (hold nuclear envelope itself up so that it
does not collapse onto the matrix) it is found inside the nucleus holding it up from the inside it lines up
inside the inner membrane of the nucleus underneath it and holding it in place
• nuclear lamina is made of three lamin proteins (a,b,c) all connect to make blanket sit underneath
nuclear envelope to give it support
• Lamin proteins are intermediate filaments so they are all about structural support. The lamina also
holds the content in various locations (Provides anchoring spot for all the chromosome, tethers and
holds onto loops of chromatin
• Since looped domains are attached to the lamina each chromosome has its own location in the nucleus,
for every chromosomes you can trace somewhere where it connects to the side of the nucleus
• nucleoplasm is inside and cytoplasm is outside
Nucleolus
• ribosome factory, dense with protein and RNA all being assembled into small and large subunits of the
ribosomes
• a lot of chromosomes anchored to one side but have loops that extend to same one region these loops
are special because they have the genes that code for the ribosomal RNA (hundreds of copies of this
gene)
• ribosomes are made of ribosomal proteins and ribosomal rnas (transcribed by pol 1) genes that code
for ribosomal rna that is what is on the loops , cells have hundreds of copies of of ribosomal rna genes
• ribosomal protein is made in the cytoplasm and are brought into the nucleus which are then assembled
into small or large subunits (all in one location jam packed filled with protein which explains why they
stain so darkly) PROTEIN & RNA & DNA
• portions of different chromosomes contain the many rRNA genes and they all loop together to form
the nucleolus
How do ribosomes get out of nucleus?
• the nuclear envelope has holes in it "nuclear pores" that go through both
membranes. huge passageways spanning between both membranes of the
nuclear envelope that provide connections, communication between the
cytoplasm and nucleus (surprisingly big 25x bigger than ribosome) ribosomes
go through nuclear pores
• Nuclear pore complex- huge passageways between cytoplasm and nucleus
they can be opened and closed (made of hundreds of proteins but plugged
with massive structure made of protein (also called nuclear porins, protein
collectively that’s part of nuclear pore)
• regulation is achieved because the pores con be closed or plugged up by
some of those cytoplasmic extensions
• small molecules that can be dissolved in water can be carried across nuclear
pore without any help (ions) since water can easily pass through (not
regulated)
• large macromolecules need help and nuclear transporters/receptors
(collection of proteins) help them since they escort messenger rna dna
proteins ribosomes macromolecules etc) bind and guide to protein then to
pore and open it
First eukaryotic receptor protein was identified by studying a virus
• sp40 infects eukaryotic cells and viral protein that gets into nucleus
• scientist took protein and infected cells with virus (created a lot of mutants of proteins that
were missing very different regions of amino acids and wondered if they all still got into
nucleus?)
• usually mutants got into the nucleus all the time but one time however a particular group of
amino acids that have been changed after infection the viral protein stayed in the cytoplasm
• amino acids removed from this protein must be required to have protein to get into the nucleus
• nuclear localization sequence - group of amino acids sequence that is needed for protein to get
into nucleus (must be some particular protein that binds to sequence and carries it to nuclear
pore and opens it up) nuclear transport protein
• Messing up this sequence will not let it bind and it will never get into the nucleus
Importin-imports proten into nucleus
Nuclear import and export
Regulatory transcription factor will always have two domains. Stretch of
amino acids that binds to DNA (DNA binding domain) and another stretch of
amino acids that allows protein to bind to another protein (protein protein
binding domain) so that it can bring in histone modifying enzymes or general
interaction with mediator
• must also have stretch of amino acids (nuclear localization signal) little
addresses if it wants to bind to importin and go into the nucleus
• importin protein is in cytoplasm and will interact with nuclear porin
which causes it to open up through the pore and out to the other sides
• import binded to cargo through the NLS Importin binds to nuclear porin
causing it to open up, now able to deliver protein to nucleus
The whole story
• nuclear protein bound to receptor came into the nucleus though nuclear pore, something lets go of
something and protein is delivered to nucleus
• Ran GTP binds to importin and causes importin to let go of receptor (its job) ran is an example of a
G protein (G protein are molecular switches, protein that can bind to either GTP or GDP) if they are
bound to GTP they are on and active doing what they are supposed to be doing. When bound to GDP
they are inactive or off, stop doing whatever it was doing (they are regulators) switch needed to
regulate
• Protein that turns on the switch is a protein that comes in takes off GDP and replaces it with GTP
(guanine EXCHANGE factor)
• GTPase takes off phosphate of GTP and instantly become GDP, G protein does this itself
• Regulation is needed because we only want him to do this when it is time to turn off the system
• Ran is only a GTPase when gap says it is (chop off phosphate and turn into ran GDP)
• ran GTP binds to ___ and causes cargo to be let go. RAN GTP causes the cargo to be let go in the
nucleus (goal of import to get transcription factor into nucleus)
• Exchanging needs to be done in cytoplasm (GAP) gap to active hydrolysis
• Exchange factor in the nucleus to cause exchange from GDP to GTP
What are some protein that would contain an NLS?
• proteins that need to get back into nucleus like polymerases,
histones, transcription factors, lamin proteins etc
Class meeting 18
April 1 2024
Prokaryotes both transcription and translation occur in the same space (cell) mRNA as it is being transcribed
begins to be translated (ribosomes jump on and start translating it). In prokaryotes the mRNA is the direct
product of transcription it contains all of the sequences in the DNA template both transcription and
translation can occur in the same place on mRNA as it is being transcribed
Eukaryotes mRNA is in the nucleus has to get out in the cytoplasm before it can get be loaded onto
ribosomes. In eukaryotes pre mRNA is the direct product of transcription and it contains all of the
sequences in the DNA template in the nucleus
Translation needs
• fully processed mRNA
• Ribosomes (small and large subunits made of rNAS and r proteins
• Fully processed charger tRNAs
all are together in the cytoplasm
Eukaryotic ribosome
• machine on which translation happens made of a a bunch of proteins and RNAs
• large subunits has 3 ribosomal RNAs (5s, 5,8S, 28S rRNA) and a ton of ribosomal protein that get
together to form large subunits
• small sub unit only has 18 S RNA that forms the small sub unit
• S28 is also an enzyme that catalyzes the central reaction (formation of peptide bonds) is often
refereed to as petidyl transferase because it transfer one amino acid to the next to make the
peptide bond
Ribosomal RNA
• RNA is made through transcription, it is transcribed in the nucleolus by pol 1 ribosomal RNA genes to
ribosomal rna
• many copies of ribosomal DNA genes because cells need ribosomes all the time and a lot of them
Why do our cells make so many ribosomal RNA genes ?
• ancestral cell had some ribosomal RNA gene and over time made ribosomes translate etc as evolution
occurred that region of genome was duplicated and over evolutionary time duplication happened
accidentally but was selected for ribosomal protein gene since it was easily made
• ribosomal proteins are transcribed on regular old genes that make messenger rna by poly 2 , they are
not clustered together and that messenger rna is sent out to cytoplasm where it is translated to
ribosomal protein and those proteins are bought back into the nucleolus this results in small and large
subunits that are shipped out through nuclear pores on their own (do not come together yet)
TRNA
• Linking molecule in nucleotide and amino acid convert messenger. Messenger rna to creation of
polypeptide
• Made through the process of transcription (transcribed by poly 3) , TRNAS are first synthesized as
pre tRNA (they have introns) that are ultimately removed nucleotides clipped off 5’ and 3’ ends
• Every CCA is added to 3’ end
• TRNAs always assumed clover leaf structure (double stranded RNA regions that are complementary)
folded into stems and loops tertiary structure . Form base pairs of RNA
every tRNA has two business ends that explain the function of the tRNA as
linking models because at one end (torch) there is an amino acid which means
language of amino acid is known
• Base is the anticodon , both link together amino acid and nucleotides
• charging tRNA is when you attach the amino acid which is needed for
translation, we need the correct amino acid
• Enzyme has to be huge to be able to look at anticodon and have a binding site
for amino acid (tryosanyl tRNA synthase) knows what anti codon is and must
bring in correct amino acid and attach it to
• This all happens in the cytoplasm and are waiting to find a messenger rna
and bring in the amino acid when needed (20 different versions of enzymes
because they must be capable of recognizing
Open reading frame - from the Aug (stop codon) to the stop codon
RNA does not start on AUG , the code has punctuation
Degenerate or redundant - more than one triplet can code for a single amino acid Degeneracy results in
the wobble hypothesis
•
The wobble hypothesis
• the first 2 nucleotides of codon must follow all complementarity rules
• however while the 3rd nucleotide of codon must follow purine: primidine rue, it does not have to
follow the # of hydrogen bonds rule
• Relatedness of all organisms code is universal language was passed down to every single offspring
The steps of translation
Initiation: get together a ribosome on a messenger rna. Happens in multiple steps where small subunits and
large subunits dont get together first. Small subunits will bind to messenger RNA first
• complex has to scan down messenger RNA to find AUG because tRNA had the anticodon
• Now large subunits comes and lines up with small subunit and is ready to initiate translation
• first tRNA binds to P site and now initiation complex is formed , next tRNA comes in lands on A site
temporarily hydrogen bonds and another amino acids is able to elongate the polypeptide
• Each new peptide bond is made by peptidyl transferase (28S rRNA)
• everybody needs to shift and get down to next triplet, ribosomes slides down p site to e site now
exits, a site to e site , new guy comes to a site
• E site is exit site, P site contains a tRNA with the growing peptide attached, a site contains a tRNA
with a single amino acid
• Ribosomes move 5’ to 3’ move on down polypeptide is made , before peptide bond they are named
correctly. Stop before translation stops. UGA is the stop codon which means that termination occurs
April 4, 2024
Class Meeting 19
Translation - many ribosomes will continue to load themselves on the mRNA (polysomes)
when it is being translated (this is constantly happening)
How do proteins get where they need to be after they are made?(Protein sorted and targeting)
• begins in the cytoplasm where all proteins sit and it all depends on where they are going
• The proteins that are needed in the cytoplasm will not move because that is where they are going to be
used (10 enzymes of glycolysis stay there, as soon as they’re made theyre not moved)
• Category 1 : Nuclear proteins (stretch of amino acids somewhere in the protein that is the nuclear
localization sequence protein bound by a protein that drags it over to the nuclear pore (importin example)
interacts with it and gets sent in to the nucleus) it has a nucleus address (structure of amino acids that
get it to the right place) bound by nuclear import receptors and dragged over to pore
• Category 2 and 3 ALL protein needs to get across the membrane out of cytoplasm into some organelle
• Category 2 Proteins are proteins that just have to cross that membrane and reach the destination
everybody who lives and works in the chloroplast mitochondria ER and peroxisome. Commute is finished
arrived at work. Ex: chloroplast and photosynthesis
• Category 3 begin journey going across membrane but have to get into a vesicle to get into their final
destination (2nd form of transport) ex : Golgi , lysosome, plasma membrane, any protein secreted.
Connected by a system of vesicles they are called one system “endomembrane system” because they
are all connected with endomembrane vesicles
• Category 2 and 3 protein is the same for all categories since they have a unique structure of amino
acids (nuclear address/ nuclear signal sequence that is bound by import protein like importin)
N
active
Mitochondrial
Signal sequence
pyruvate denydro
transport bbinding
domain
pyruvate
buding
domain
site
C
NAD binding
domain
Mitochondria
• mitochondria signal sequence recognized by mitochondrial import protein
• Proteins from cytoplasm into matrix must cross two membranes outer and inner.
Protein goes through the porin channel (big channels that are non selective) but then to
get into matrix pyruvate dehydrogenase transport binding domain is needed (need to
worry about binding domains to get across selective channel) now it is in mitochondrial
matrix woo *domains needed to do its job and domain needed to know where it is going
• in the cytoplasm pool of large and small ribosomal subunits messenger rna arrives in
cytoplasm and loaded on to free ribosomes that load onto rna and protein is produced
and sent to mitochondrial chloroplast etc
Who is not synthesized on free ribosomes
• all the proteins from the endomembrane system ex: aquaporin, calcium channels
• Translation does start on a ribosome floating around in the cytoplasm but if it is destined for
endomembrane system just after translation begins it stops (or slows down) and this is because a signal
sequence is emerging as soon as translation has got this far and asks to be taken to the ER (SRP signal
recognition particle) = ER transport protein SRP binds to signal sequence and gives it time to get dragged
to ER till translation resumes. Ribosomes stuck to ER after and is called a membrane bound ribosome
How do (soluble) proteins get into ER ? Ex : acetylcholine (live in the lumen)
• all protein that works in the ER or other organelles outside the cell first
are found in the ER (fully synthesized proteins)
• mRNA ribosome protein begins to translate first nucleotides emerge and
ER signal sequence binds to SRP, when SRP binds translation pauses and
when it continues protein can be threaded right into ER.. This must be
translated and translocated at the same time .
• It is dragged over to surface of ER that anchors everything to the
protein translocator sitting in membrane of the ER (SRP allows this)
• SRP does its job after bringing it over and disappears (translation starts
up again) Protein can be threaded through the protein translocator and
into the ER
• ER signal sequence is removed b/c it is not needed anymore, must be
first domain at end terminus
How do integral membrane proteins get to the endomembrane system ?
• story begins exactly the same way in the beginning protein would have ER signal
sequence bound by SRP and brought over to ER
• Still being translated add on some ribosomes start threading that protein
• If it is an integral membrane protein as it is being threaded at a certain point part
of this protein will be apart of every single transmembrane protein must have a
region that is there (transmembrane domain)
• Translocation stops because right behind transmembrane domain (non polar
stretch of amino acids) there is a special sequence of amino acids called the stop
transfer sequence, it tends to be very polar and stops it from moving on. Now
that protein can stay in translocaton until the sides door of the translocon to
open up (periodically open and close)
• When they are open hydrophobic tails of phospholipid come into contact with
hydrophobic region of protein which allows it to slide right into the membrane
• this is true for any protein that lives or works in the endomembrane system
(including plasma membrane proteins)
• Na+/K+Protein is in membrane that becomes the vesicle to move them along and
fuses with organelle
Vesicle Transport
• soluble protein going through the system made in ER, folded and
has sugars attached. Put into vesicle get into golgi then another vesicle
and end up in another destination endosome or secreted outside the
cell
• Integral put into membrane right in the ER would end up in a
vesicle if meant to be in plasma membrane then the Golgi another
vesicle then set out till it is sitting in the plasma membrane
• proteins end up being moved through because it ends up in
solution where the vesicles is and starts forming around them (non
selective bulk transport)
• Selective mechanism (protein has to get to Golgi or plasma or
secreted) targeted mechanism that has sequence that needs to be
targeted
Vesicle Transport
• secreted protein has to go the furthest, scientist followed cells producing this peptide hormone
• Smooth er had no ribosomes attached (membrane made here), Rough ER has ribosomes on it. Protein
gets into rough ER and about 20 minutes later they end up in Golgi apparatus and then to secretary
vesicles where some of it starts to be secreted
• In order to function some proteins are not yet ready to function so they must go a longer route
• Every protein that sticks out of the cell has some sugar attached to it
• In the ER (rough er) protein has to be folded correctly special chaperone proteins helps fold any
protein coming into the ER. Sequences are chopped off in the ER and some of the sugars are also put
on the ER. (protein folding cleavage, initial glycosylation) chaperone proteins aid in this. Both ER and Golgi
(candy stores)
• Protein will end up in a vesicle pinched off and will go fuse with the Golgi apparatus (which has two
sides). A bunch of membrane compartments that are flattened pancakes cis face faces the ER
(further glycosuylation, phosphorylation) fuses with it until it gets to the trans face (faces to plasma)
and will sort out proteins. Proteins that leave the trans Golgi network are sorted into the correct
vesicle and off they go. To lysosome, endoscope, plasma membrane or outside the cell (Train station
and bakery)
How are these vesicles made ?
• new group of proteins that make these vesicles (coat proteins). three different type that live in
cytoplasm and binding to cytoplasmic surface of the membrane causing a bubble to form (Clathrin,
COPI,COPII)
• Membrane cytoplasm is white lumen is gray red protein needs to be secreted will bound to specific
receptor in membrane and grab what you want to secrete. COP proteins will coat outside surface
of region of membrane they are very rigid and snap together that are cup shaped. They form a
little ball scooping up bit of membrane into ball
• Claithrin coated vesicles needs adaptin which binds to cytoplasmic domain (stiff cup shaped proteins
that snap together and form a ball pinching a bit of membrane). Protein is now captured and
dynamin pinches off last bit of membrane which forms the vesicle (three different groups only
difference is where they operate)
• Claithrin hangs out in cytoplasm between golgi and plasma membrane
• COP between ER and golgi ,
• COP 1 vesicle brings vesicles back from Golgi to er (retrograde) moving backwards
• COP 2 from ER to Golgi anterograde from middle of cell to out
Class Meeting 20 April 8, 2024
Termination of translation (review)
• ribosome slides around messenger rna slide into next triplet nad it one of the stop codons so there is no
tRNA to come and bind it does not exist
• When this happens a release factors bind to the A site. Can bind to one of the three stop codons
(signal that everything is over). Now chop off polypeptide chain by peptidyl transferase (28 S) and
everything disassembles
Why do vesicles go in the retrograde direction ?
• needed to retrieve membrane so that the ER does not disappear and all convert into vesicles
• also retrieve accidental travelers (protein that is supposed to live and work in the ER and ends in
a vesicle it is going to be taken out to the Golgi ) that are supposed to sustain in the ER. First
example of targeting proteins to vesicles (COP 1 vesicle proteins that function in ER and
accidentally end up in COP 2 vesicles and end up going)
Target proteins specifically to vesicles
• proteins that function in the ER in a cop 1 vesicle and end up in the COP 2 vesicle
which means Golgi when they are supposed to function in ER we only want to
bring this back so we need ER address and states it does not belong there.
Targeting protein is a membrane receptor that binds to receptor protein so that
they will get into vesicles and be brought back (selectively bring in back protein
that are supposed to live in the ER)
• The sequence that is recognized is lysine kDel four amino acids are present in
any protein that is what is recognized by receptor. Membrane bound protein
binds (KDel receptor) to any protein with that same sequence in its primary
amino acid sequence and receptors cytoplasmic side binds to COP 1 vesicle
Proteins targeted in the lysosome (live and work here)
• lysosome is a recycling center (for cellular debris and old organelles). Old proteins organelles etc are
broken down by acid hydrolases to their smallest building blocks and then sent back out to the cell.
• Nutrients also get sent to the lysosome (pantry) get distributed to the cell or can be broken up and
then sent out to the cell. A lot of stuff brought in are nutrients that the cells need.
• Proteins that live and work in the lysosome are enzymes (acid hydrolyases) these are the proteins we
need to target to the lysosome
Example 2 : transport of lysosomal enzymes to the lysosome (specific targeting of a protein to a vesicle)
• starts with fully synthesized acid hydroysases found in the rough ER (category 3)
• Lysosomal enzymes has stretch of amino acids recognizes the sugar to this acid hydrolyases.
Particular amino acid sequence that says “take me to the lysosome” in the case of lysosomal enzymes
there is one stretch of amino acids and the receptor that recognizes the sequence actually
recognizes the sugar that is added by glycosylase (sugar is called mannose) and attaches it to the
sequence (amino acid is still needed though since address is needed for sugar to be put on protein)
• after it is glycoslated it heads out to the Golgi and in the Golgi the mannose gets phosphorylated. IN the
Golgi there is a kinase that adds phosphate to mannose it is attached to the 6 carbon on mannose
• now it can bind to receptor….. Clathrin is binding to outside part of protein sticking to the cytoplasm
is where adaptin and clathrin will be.
SNARE protein
• something in toxin prevents signaling from neuron and muscle cell
can cause paralysis
• The protease target is the snare protein (without snare protein
which is responsible for vesicles finding the right membrane and
fusing with them). Snare proteins are needed for signals since the
receptors will stay in their vesicles and not signal without snare.
• SNARE proteins come in pairs (two versions based on their
location). One found in membrane of the vesicle and the other in
the —
• Acid hydrolyases only work in the lysosome (acidic conditions) pH of
lysosome is 4.6 so enzymes can only work at this pH. When they
travel through other fluids with pH lower than 7 they are inactive
Mutations
• COP II (ER to Golgi) Will be stuck in the ER bc it cannot get into a Cop 2 vesicle or it will be unaffected
• VSNARE in the vesicle formed at TGN vesicle will not have snare protein, will be free floating
• COP I are for retrieval of ER proteins moving lysosomal proteins should not be affected
• Adaptin stuck in the TGN and will never make vesicles that is targeted to the lysosomes
Cells bringing things in by endocytosis (opposite of exocytosis)
• at plasma membrane clathrin coated vesicles are formed pinched off into the inside and brought into
the cell. Endo - into the cell inside a vesicle.
• Vesicles will be formed at the plasma membrane and will meet first organelle called endosome first
stop and (organelle thats only job is to sort) in the endosome there are two designations either stuff
will be targeted to lysosome or from the endosome a vesicle could go back to plasma membrane.
Sorting helps separated everything brought into the cells vs things that can be brought back to
plasma membrane
• Next stop is the lysosome (key organelle for vesicles inside the cell to destroy things but also for
everything coming into the cells)
• everything that goes to the lysosome (good nutrients like lipids and water) and the bad (pathogens)
Three categories of endocytosis
• phagocytosis in humans only done by macrophages, vesicles are HUGE
compared to endocytosis. Cell is able to extend bits of membrane that it
wants to engulf (pseudopods) fuse at the top. Macrophages can do this
because they can make the pseudopods made by actin microfilaments
that and push further
• Pinocytosis - non selective small molecules and water (bits of membrane
pinched off all the time) constitutive process does not turn off or on
• Receptor mediated endocytosis - something floating by then bound to
receptor and is selective for cargo (protein and nutrients) cell can
choose what it is going to eat. If binds to receptor is going to get stuck
there long enough to bind into a vesicle
Cells wanting to bring into cholesterol
• brought in by the LDL particle (all of our cells need cholesterol to make our membrane but cells cannot
make cholesterol so it has to be grabbed from our diet) if cholesterol is not bought into cells it stays in
artery and veins (blood vessels) and causes heart attacks by forming plaques
• LDL particle carries cholestrol around the blood (cholestrol is highly hydrophobic its a lipid) needs to be
transported through blood stream in aqueous solution. (Low density L protein particle) LDL particle are
made out of membrane lipids, monolayer of phospholipids. Entire inside is hydrophobic so it can be
stuffed with cholesterol
• ApoB receptor (protein protein interaction)
• grey stuff is extraceullar fuild , cell out a bunch of ldl receptor in plasma membrane cell is ready to take
up cholesterol if any particles float around and bind to receptor
• extracellular domain has bonding site for LDL, cytoplasmic domain clathrin is on the outer region and
operates in both directions so adaptin binds to them and clathrin as well
• Because membranes are fluid we can end up with a unch of those guy as clathrin snaps together until
a pit is formed (clathrin coated pit)
• Pulled off enough membrane to make up a dynamin comes along and pinches off and vesicle is brought
into cell
• clathrin spontaneously comes off and disassembles as soon as vesicle is done being made without
clathrin now we have membrane as long as we have right snares around it will fuse with endosome
• Endosome is where sorting of things brought in by endocytosis, in particular pH in endosome is a little
more acidic then extracellular fluid which causes receptors to let go of their ligands
• Ligands end up in vesicles going to the lysosome, receptors end up in vesicles getting targeted back to
the plasma membrane. Cell just recycles the receptor
Regulatory transcription factor
• enhancer dna binding domain some part
of protein that will bind to DNA
• Protein binding domain bring in mediator
histone modifying enzymes
Class Meeting 21 April 11
• all biological processes happen in aqueous solutions “baggies of water” inside a eukaryotic cell
contents are localized / being moved to particular locations
Cytoskeleton (unique to eukaryotic cells)
• water plus cytoskeleton. Cytoskeleton is a network of fibers present in every eukaryotic cells that
provides structure to the cell (structural support and 3D shape) prevents it from collapsing
• The cytoskeleton also organizes and localizes contents of cell (organelles and macromolecules)
• is is very flexible and can be altered in various way (change structure)
Three fibers found in all cells
• microtubules (largest in diameter hollow tubes)
• Micro filament (smallest in diameter 7,8 nm) solid fibers
• Intermediate filaments intermediate in diameter between microtubules and microfilaments
• Intermediate filaments provide structure and support (bones of cells cytoskeleton), and resistance
to strength of pressure/stress is applied.
• Different cell types turn on different intermediate filament genes (form determines function). The
expression is tissue specific and allows intermediate filaments to look different. Together provide
different function of cytoskeleton
Intermediate Filaments
Epithelia cells - keratin filaments
Connected tissue and muscle cells and glial cells - vimentin
Nerve cells - neurofilaments
*one exception is nuclear lamins which are found in all animal cells
• intermediate filaments are made by beginning with proteins that are all
very similar to the naked eye. Protein itself 3D shape is a fiber and
are coiled around each other to make a dimer in the same orientation.
Two dimers coiled together will be anti parallel when coiled. Two ends
identical and a thick fiber forms
How to assemble an intermediate filament
• made in various location in the cell (wherever they are needed)
• Add in some IFAP’s (IF associated proteins) connect them to each other and for example connect
them to the membrane put them in their right location to create whatever architecture needed for
that cell this results in 3D cellular shapes
Microtubules and Microfialments
• critical for structural support but have additional roles in movement either inside the cell or the movement
of the cell itself
Microtubules
• largest hollow tubes (all identical no tissue specific) begin with alpha tubulin, they are all made of this
• Made of tubulanes (alpha and beta) alpha and beta get together to form heterodimer ( protein is
slightly different) get lined up end to end to form long fiber (protofilament) it has two different
ends one end has all betas t the other are all alphas.13 of these are wrapped around to form a
tube that surround a central cavity One end has all betas the other one has all alphas. Structural
polarity - structurally two ends are different and you can tell them apart
• Intermediate filaments do NOT have structural polarity
Microtubules continued
• can grow and shrink,they can be stable or unstable inside the cell, this has to do with the
difference in two end, tend to grow at beta ends (+ end) heterodimers are added will join and
happen at top. They will shrink at (-) end, not growing or it can be shrinking, heterodimers could be
coming off bottom end where alphas are (Nothing to do with charge)
Microtubules display dynamic instability
• allows cells to assemble and disassemble very quickly if necessary
• Separation of chromosomes the structure that does it is made of
microtubules, very important that call can disassemble and reassembled
the spindle microtubules
• Everything is regulated by the cell whether anything is growing or
shrinking , regulation is dependent on beta tubulin is a G protein (GTP) will
do whatever it’s supposed to do (GDP) stops whatever it is doing
• When we need to add dimeres we are going to add them with beta
being binded to GTP high affinity for other heterodimers (GTP CAP)
• G proteins can hydrolyze their own GTP to GDP (they are all GTPase)
• adding more to to the end at some point it will hydrolyze GTP to GDP.
• GDP has low affinity and at the end they begin to fall off (-). End of fiber
does not have high enough affinity to stay on (will not fall off in middle)
Regulation of MT assembly and disassembly
• concentration of GTP tubulin means plus end is not growing and eventually beta tubulin GTPase activity
everything will be hydrolyzed. Complete disassembly of microtubules (decrease of GTP tubulin) both
minus end and plus end. Cells will regulate how much GTP tubulin is available to bind to GTP.
• Microtubules associating proteins (MAPS) some will bind to plus end other minus end many different
roles of proteins to influence assembly or disassembly
• Change in calcium concentration (inside cell calcium concentration is low because of calcium pASE).
Microtubules are typically stable and high calcium concentration channel floods and causes microtubules
to disassemble.
Where are microtubules formed ?
• microtubules are all born in a microtubule organizing
center (MTOC). Most common organizing center is
called centrosome (location where they are assembled)
sits right outside the nucleus (basal body of the cilium/
flagellum is also a place where microtubules are made)
in animals cells collection of protein associate with
nuclear envelope which is microtubule organizing
center in most plant cells.
• most cases MTOC in center of cell. The minus ends are
anchored in the centrosome (blocked) , plus ends are
going out of centrosome to grow out and fill the rest
of the space (plasma membrane)
Microtubule functions
• support structure and intracellular movement. The axons of neurons have
intermediate filaments and bundles of microtubules hold up the axon.
Microtubules make the spindles.
• Microtubules are central to structure of flagella/cilia. To make a flagella or
cilia you need to make microtubules (cilia lot of them and short flagella one
or two of them and they are long) same structure at cellular level.
• Micotrubuels are critical for moving things inside the cell since they are the
highways of the cell.
• Cross section through a cilium or flagellum extension of membrane is filled
with microtubules there 9 doublets on the outside and 2 doublets on the
inside, this arrangement is found in flagella and cilium this is the axoneme
(the structure).
Intracellular movement
• Microtubules are the primary highways and motor proteins
hop onto microtubules and walk along them by carrying stuff
(ubers)
• two kind of motor proteins that can bind to MT one goes to +
(kinesin) direction the other the - direction (dynein) walk one
step at a time hydrolyzing ATP molecule
• both are made of four polypeptide two heavy chains (fibrous
domain that form the dimer and globular head domain binds to
microtubule and ATP) walk on their heads and two light chains
(blue) they choose cargo what is brought in what direction
How do cilia and flagella move?
• inside axoneme motor protein is present called ciliary dynein
many of these motors line up along one microtubule and bind to
other microtubules cargo and move relatively to each other.
Dynein goes plus to minus end kinesis minus to plus end
• This is coordinated around the circle go one after the other the
entire microtubules is bent and snapped back
Microfilaments
• 7-8 nm long fibers and not hollow they are the thinnest of all
• microfilaments in every cell type are identical and not tissue specific, microfilaments are made out of
same protein (actin) globular protein sitting in solution (globular actin) Microfilaments need to line up a
bunch of these guys end to end no dimers (monomers) and twist two fibers together and boom
microfilaments. F actin - a microfilament
• MF can grow and shrink at different ends and adding to end needs to have ATP bound to that actin
high affinity for other actins and can grow at one end and shrink at the other. Bound to GDP low
affinity for other actins
• Microfilaments do not display dynamic instability they are much more stable can be grown and
disassembled easier than intermediate filaments but are way more stable. This is regulated by
microfilament associating proteins (MFAPs) that associate with them
April 15
Class Meeting 22
Microfilaments
• microfilaments are also key for structure and support, provide structure and support for sub cellular
structures.
• Pseudopods - macrophages can form by extending microfilaments surrounded by membrane and
surrounding things they want to take into the cell found in lots of prokaryotes like amoeba ,
microfilaments are important for particular sub cellular structure pseudopod
Microvilli in intestinal cells
• Apical surface of intestinal cells has a surface to find by membrane extensions and each membrane
extension is held up by bundle of microfilaments
·
Microtubules are main highway of the cell but microfilaments can be
used as side streets (motor proteins also move on the microfilaments).
Myosin is another motor protein (found inside cells myosin type v it
carries cargo) all myosin’s are motor proteins.
Muscle cells can move and contract is because of myosin and actin
reaction with one another
Myosin - has two heavy chains coiled together by tails to form dimer,
head walks around the microfilament it binds to actin which means head
region has ATPase activity (how it walks) always walk towards plus end
of microfilaments
·
Myosin Type II- similar except the tails of myosin heavy chain do not bind
cargo they twist around each other and then a bunch of them twist around a
bunch of more myosin tails (facing the other way). Coils whole different group
of myosin facing a different direction , tons of heads extend way out in both
directions. Myosin walks towards the plus end (outside) forms thick fiber
interacting with two thinner microfilament fibers. As they walk they pull actin
in towards center by the motion of the myosin heads (f actin =microfilament)
Muscle cell differentiation
• start off with embryonic cells we call it myoblast because it will
grow to be a muscle, so that means itinherits proteins that tell it it
will be a muscle (regulatory factors) then they would tell the cell to
proliferate (divide) and then the cells will fuse, one cell with many
nuclei (syncytium)
• over time long stretched out cell (muscle fiber) will form due to the
fact that these cells turn on actin and myosin genes. These
proteins get together in fibers and fill the entire contents of cell
pushing nuclei out to the side.
• myosin is in really dark region in the middle and actin is really light
region the thin filaments (microfilaments)
• sarcomere-each myosin thick fiber with two thin fibers it is binding
to on each side. Line up end to end to make one long massive fiber
Big thick stripe in middle is a end myosin
At ends of each thinner but darker line is called z line *disk*
Light stripes is called I band which is actin
• within dark band the absolute darker region is where actin
filaments and microfilaments overlap “overlap region” they interact
with one another. Heads are binding to microfilaments
• Plus ends are in the actin are in the Z disk, as myosin heads walk
towards plus end they are walking towards the Z disk. Two
microfilaments attach to z disk in plus ends and bind to myosin in
the center
• Myosin heads grab pull and let go when done in two directions they
will bring actin filaments to the center
• Relaxed muscle if contracted z disk are now closer to each other
and now there is a large region of overlap (cross bridging cycle)
Cross bridging cycle
• myosin head bound to actin and then in comes ATP binds to myosin head which causes myosin to
let go. Myosin is a ATPase so it hydrolyze ATP and a little bit of energy bends the myosin head to
new location plus end of actin. Attach a little further along to plus end but now it needs to move
back. When phosphate is released after bending now ADP is released and rest of energy is
released. Microfilament is now moved along a tiny bit. Now it needs to move more to plus end so it
a bends a little bit to plus end on the actin and then attaches again
• Now it must bend back while holding onto the actin (force generating power) dragging actin along
towards center of sacromere. The end of the actin filament is a little further towards center of
sarcomere. This must happen over, over again to contract the muscle. (requires a lot of energy).
• Bending of the head the first time is easy because the head is just moving but the second time
acting is needed to be dragged (power stroke)
• At end of cycle myosin is bound to actin as long as another ATP comes along everything repeats
myosin head is bound to actin binding of ATP causes the release
• myosin will still be attached and grabbed onto actin they will not be relaxed (rigor mortis) when
tissue runs out of ATP it will be stuck in this position FOREVER (this process needs to be regulated)
they can be relaxed by separate set of proteins who will interrupt this cycle and can determine
when myosin and actin will interact
Strian muscle
• Strian muscle actin linked regulation system involves two proteins that bind to actin (troponin and
tropomyosin)
• Tropomyosin lays along actin fiber end to end. Troponin complex is a bunch of protein that also bind to
troponin and actin.
• Interruption of myosin and actin must be interrupted so muscle can be relaxed. Tropomyosin is being
pushed over the site by tropomyosin over to cover the myosin binding sites on actin. Where myosin
would like to bind to actin. Troponin stops pushing tropomyosin so it will slides off myosin binding sites.
• CALCIUM LEVELS : troponin is another protein that can mediate different calcium levels and do
different things based if it is bound to calcium or not. Troponin is a calmodulin (any protein that can bind
calcium and change conformation) in one conformation does one thing in another it does something else.
It provides link between calcium concentration of the cell and ____
• At low calcium muscle cell is relaxed and cause calcium to go up to cause contraction but in general
keeps calcium low to prevent contraction
• As calcium goes up Troponin binds to calcium lets go of tropomyosin and it slides off the myosin binding
site and cross bridging occurs as long as ATP is available
• Troponin is not bound to calcium will push it over the site.
What are the relative concentration of calcium for all eukaryotic cells?
• signal from a nerve in contact with muscle needs to cause calcium levels to increase. Calcium levels
are typically high outside of the cell and low inside the cell. Calcium ATPase allows this since it is
constantly pumping away
• One of organelles also has calcium ATPase also high in the lumen of the ER since the ATPase is
located here. Cells can regulate how much calcium is in the ER Calcium channels are in plasma
membrane that can be opened up as well as the ER. Temporarily open gates calcium will flood into
certain region and cause something to happen.
Skeletal muscle
• filled with myofibirl so a we need a lot of calcium to regulate troponin sitting on actin filaments
• Tons of expanded ER in muscle cells (therefore filled with Calcium) sarcoplasmic reticulum so we can
flood entire cell with calcium if channels are opened. Nerve will stimulate the muscle and tell it is time
to contract.
• Neuromuscular junction nerve gets the signal when it gets to terminus sitting next to muscle cell will
release neurotransmitter and it will go to plasma membrane of muscle cell and when acetylcholine
comes along and binds to receptor sodium will flood into this cell. It will depolarize the membrane
more positive inside then the outside results needs to be that muscle cell will begin to flood calcium,
this will happen
Depolarizing membrane
• two calcium channels will open one in plasma membrane and one in ER. Plasma membrane opens voltage
gated calcium channel in plasma membrane which then pulls on the mechanically gated calcium channel in S
membrane.. tons of calcium from outside and ER since it is jam packed, contraction occurs.. sodium increas
which leads to calcium increase
• In muscle cells plasma membrane dips way into cell and brings channels right next to each other (T tubules
Class meeting 23
April 18
• all stuff comes from the cell, cells make the stuff and secrete it. Create their own environment it
must be deposited primarily by exocytosis
Matrix (Cell Wall) and (Animal Cell)
• create the structure of the tissue and organs (support) to the organism at the multicellular level
• These matrixes create the environment outside the cell we need an aqueous environment surround the
cell
Cell Wall
• tissues need tremendous support usually rigid, movement not needed and this is provided by the cell wall
• since cells cannot move around the cell wall must provide protection from abiotic (wind, rain) /biotic
stressors (herbivorous)
Cell wall is made of
• main component is polysaccharide called cellulose
(sugar)primary component of cell wall both primary and
secondary cell wall in both cases it is unbranched it is the
most abundant
• chain of glucose, long unbranched chain that allows them to
line up next to each other tightly packed together (light up
next to each other parallel that will result in a strong fiber).
• sugars can be branched branch of monosaccharides.
Sugars have hyrdoxyl groups and are very polar (polar
interactions) so they are highly hydrophilic , structure of
sugars in cell wall draws water in. This results in an
aqueous environment for stress and travel.
• in mature cell walls there is lignin - a massive and very rigid
organic molecule in secondary cell walls – the main
component of “wood” it is deposited into the cell wall as
plant matures
• glucose molecules are strung together and is made exactly
where it is needed, cellulose is made on outside surface of
cell. This is done by an enyzme that strings together all
glucose molecules that is an integral membrane protein
found in the plasma membrane. The active site of this
enzyme is on the outer side so as glucose floats by the
enzymes grab glucose and link them together to form an
actual fiber (multiple enzymes called rosettes) catalytic
domain is on the external surface, cellulose is in perfectly
straight lines. Rosettes can cruise down the membrane and
lay down cellulose as they go since the membrane is fluid
• Rosettes are going down in straight lines
ECM of connective tissue is always ECM then cells. Everyone else is mostly cells
Extracellular matrix
• animal cells can move around and different cell types with
different behaviors tremendous diversity
• fluid bone is also extracellular matrix but is highly rigid
• connective tissue vs everyone else
• collagen is the main component of both kinds of ECM,
tremendous diversity within the collagen family (Found
outside the cell) multiple different genes that code for
slightly different collagen.Any given cell will only use one kind
because there is a slightly different protein. Different kinds
of collagen in different parts of the tissue.
• cellulose and collagen provides similar roles but there is a
difference since collagen is a protein and is made inside the
cell on a ribosome is must be synthesized inside the cell and
then deposited by exocytosis. collagen is syntheses on
membrane bound ribosomes in ER. Must have ER signal
sequence etc
• three of them are coiled together into a triple helix (pro
collagen) . The loose ends are chopped off so that they can
bind tightly together to create thick fiber that is also bendable.
• Enzyme clips them off and assemble into huge collagen fiber
this occurs after secretion this happen in the outer cellular
matrix.
Connective Tissue
• all connective tissue most of the mass is just extracellular matrix the rest of it is just cells making that
ECM
• Osteoblast (bone) is a cell that lives in and secretes the cartilage in bone. Fibroblast live and secrete
tissues in tendon and the eye.
• Functions : holds different parts of organism together (essential role) also important in resistisnt
mechanical stress on the organism. Different function depending on the tissue. These are the tissues in
the multicellular organism that connects everything together.
FIbronectin
• second protein found in connective tissue (alongside collagen). No protein
can grab onto collagen but cells have receptors for fibronectin (link
between cells and collagen
• At the end terminus the ER signal sequence is already chopped off.
Quaternary structure because of multiple polypeptides joined by Sistine
amino acids
• Extracellular matrix is more harsh environment than the cytoplasm of
the cell. Disulfide bridges will be used . Interaction with lots and lots of
weak reactions there are no covalent bonds instead multiple
polypeptide held together by disulfide bonds (lives outside of the cell)
• Connective tissue is air bags of organisms to resist stress it is filled
with water (proteoglycans draw water in) massive aggregate mostly
sugar with a little bit of protein.
•
•
•
vesicles containing proteogylacans fuse with plasma membrane
(polyspemry is prevented) sperm contacts other membrane and
causes vesicles to fuse and dump the proteoglycans between the
space and water will eventually follow. Water flood into it causes
egg to lift from surface
Trigger is when sperm contacts the egg this causes release of
calcium inside the egg cell. Causes cell vesicle fusion.
Proteoglycans are mostly sugar with a little bit of protein (huge
macrmolecular complexes) you start with huge long fiber (Central
core of it) carbohydrate (GAG) attached to central polymer is
multiple smaller proteoglycans attached (protein but mostly sugar)
hence they bring water so it it a fluid gel not flat like surface this
creates (water bags) to resist pressure and force. Also provides
movement of molecules diffusion of small molecules. It it also filled
with aggregates that prevents the movement of really large things
like pathogen
ECM of everyone else
• epithelium (ex) intensine. Epithelial tissue is a
connected sheet of identical cells. Two sides
(apical) and (basal)
• With all sheets apical surface is either facing
water or air in the gut it is facing the fluid of
the lumen inside the gut.
• no extracellular matrix at the top but there is
one at the bottom called the basal lamina (thin
flat sheet) where collagen is.
• Two more proteins involved integrin( (cell
service receptors priced by cell to hold on to
laminin to hold onto collagen) and laminin (plays
exact same role as fibronectin just in
different location)
Cell junctions
• protein complexes found between cells that have variety of functions
• tight junctions are only found between cells of an epithelial sheet.
Tight junction seals neighboring cells together in an epithelial sheet to
prevent leaked of molecules between them. It is a barrier. No water
can get between those cells create true sealed barrier. Contents of
guts dont leak into rest of body and bodily fluids dont leak out of you.
Only some small ions can get through but no fluids can. This is made
by grands of occludin and claudin proteins
• Prevent movement of integral membranes and keep them where
they are supposed o be living
Junctions continued
• adherens junctions (attached to microfilaments) and desmosome provide mechanical support
• The adherens junction joints as an actin bundle is one cell to a similar bundle in a neighboring cell
• The desmosome joints the intermediate filaments in one cell to those in a neighbor
• Snaps holding in together the cell the integral membrane protein is the same kind of protein
(Cadherins) calcium dependent adhering proteins which means they need to have calcium bond in
order to interact with one another
• Each cadherin will only bound to the same cadherin (everyone will sort out) e cadherin to e cadherin
and end up with two different tissues (tissue specific expression)
• Expression of cadherins are essential to epithelial cells (expression of different cadherin leads to cell
sorting)
• desmosome attached to intermediate filaments and adherens junction are attached to microfilaments
• Gap junction is when many proteins creating big channel that lines up perfectly with channel in
another channel lines up perfectly with channel in membrane next to it so cytoplasm can allow small
water soluble molecules including ions to pass from cell to cell (special cases where cells have to be
metabolically coupled)
• Ring canals cytoplasmic ridges between nursery cells and oocyte
• In plant cells we do not need mechanical support, any cells sealed together to prevent leakage, but
we do need a gap junction , times where plants cells are connected by their cytoplasms to
coordinate metabolic function but structure looks different (plasma desmosome) same thing as gap
junction
• Poke hole through cell well and membrane of each cell line the hole so that two cells can share
cytoplasm and often share endoplasmic reticulum
Class meeting 24
How do cells duplicate themselves?
Cell Cycle
• life of a cell from when its born at mitosis until it goes through another mitosis
• Interphase is everything except mitosis
April 22
G1 (45 mins)
• period of time where cell must become twice its size by duplicating all of its contents and then divide
• essentially all is duplicated except DNA (cell waits to duplicate its DNA) cell wants to ensure it doesn’t
duplicate its DNA because it is the single most intensive energy thing a cell must do so it must know
everything else is ready for cell division
S phase (6-8 hour)
• DNA replication occurs
G2
• set up mitotic spindle and allows cell to enter mitosis . Are we ready?
The cytoskeleton during mitosis
• mitotic spindle is made of microtubules( separates the chromosomes) Cell duplicates it chromosomes
and duplicates centrosomes. Two centrosomes move to opposite poles of cell and make new
microtubules.
• Microtubules orient chromosomes line up to end. Minus ends in microtubules are in centrosomes plus
ends face center of spindle
• goal is to separate chromosomes as microtubules at plus end start to depolymerase pulls
chromosomes towards pole and pulls them apart
• microfilament form circular structure around middle and contract
Why would a cell decide to divide?
• cells have to be told to divide
• Newly born cell can continue to divide or decide not to divide (exit the cell cycle)
• cell can withdraw permanently and differentiate (turn into neuron muscle cell etc ) cell cannot divide and
differentiate at the same time
• some cells can enter G0 quiescent not dividing and not differentiating
• different environmental cues that will tell them what to do, cell can go through apotosis
Cell that will continue to divide
• activate a series of regulatory switches inside the cell
• progression through every stage requires the activation of one of these
switches
• helicopter parents (checkpoints) end of gl to go into sphase and end of
G2 to go into m phase
• point of no return (can not unreplicate DNA)
• G2 checkpoint is entice because cell can pause if necessary. Cell may be
able to fix problem
• every molecular switch is made of two proteins (kinase that
phosphorylates ) and cyclin . cyclin dependent kinase
Cyclin has to be gone in order for
Ul attaches ubiquity to target protein