Midterm No. 1 Flashcards
Smallest objects seen in a traditional light microscope:
Large virus
Smallest objects seen in a super-resolution light microscope
Nucleosomes
Smallest objects seen in an electron microscope
Large/heavy atoms
Smallest objects seen in x-ray crystallography
Nucleotides
During the synthesis of a protein (linking of amino acids), is water a reactant or a product of the chemical reaction involved in creating peptide bonds?
Water is a product. Peptide bond formation requires condensation, which produces a water molecule
During the breakdown of a protein into individual amino acids (a reaction that is also referred to as hydrolysis or digestion) -is water a reactant or product of the chemical reaction?
Water is a reactant. The breaking of the peptide bond requires hydrolysis, which uses water as a reactant.
Cysteine
Can form covalent disulfide bonds
Glycine
Smallest AA, single hydrogen
Proline
Generates kinks in AA chains, makes them incompatible with secondary structures
If you look “down the barrel” of an alpha helix, where are the R groups?
The R groups face outwards of the alpha helix
True or false: The individual strands that make up a beta sheet can come from non-contiguous regions of the primary structure.
False. The individual strands are contiguous, it’s only the between regions of the strands that could be non-contiguous
Where do “domains” fit in–relative to primary, secondary, and tertiary structure?
Domains are modules of tertiary structure, aka sections of folded tertiary structure made up of secondary structures like alpha helices and beta pleated sheets
How do “genetic innovations” relate to either the evolution of protein families or the protein relationships?
Genetic innovations relate to the evolution of protein families by generating new protein variants in the family, through intragenic mutation, gene duplication allowing one gene copy to diverge, or DNA segment shuffling. By studying the similarities, or conserved aspects of folded structure and AA sequence, we can learn more about the protein’s function and evolutionary history.
Individual protein domains are formed from a contiguous part of the polypeptide. Why is that relevant (and useful) for scientists who might want to genetically engineer a novel hybrid protein?
Altering the contiguous part of their hybrid protein will alter and potentially deactivate the domains.
Altering the non-contiguous part of their hybrid protein may alter how the protein folds.
Intrinsically disordered proteins (or intrinsically disordered protein domains) have more ____ amino acids and fewer _____amino acids. How does this finding relate to the oil drop model of tertiary structure from the previous lecture?
IDRs typically have more polar or charged AAs and fewer nonpolar and uncharged AAS. This means that IDRs will typically be found on the exterior of proteins configured in an oil drop model, where they can interact with the nearby water molecules and away from the interior hydrophobic AA residues.
Also, some ID proteins will form liquid condensates with other proteins that have multiple binding domains. These liquid condensates behave like liquid droplets, similar to the oil drop model. Some are considered to be membraneless organelles.
What are some ways that intrinsically disordered proteins function within cells?
Histone tails are an example of IDRs within the histone proteins
IDRs in liquid condensates are biologically important as stress granules and in germline cell determination
Liquid condensates
Condensates are formed by liquid-liquid phase separation, and are involved in stress granules and germline cell determination. Conditions like protein concentrations, post-translational modifications, ion concentrations, and temperature affect liquid condensate formation.
What is an amyloid? Why is it relevant to a lecture on protein folding and protein assemblages? What is a “cross beta sheet”? How is it relevant to Alzheimer’s Disease and Parkinson’s Disease?
Amyloids are plaques formed by inappropriate protein aggregation that cause Alzheimer’s. They form when improperly folded proteins stick together, usually via exposed hydrophobic AA residues
Cross beta sheets are the smaller subunits that make up the structure of the amyloid fibril. These are organized, structured aggregates that assemble into the thicker amyloid fibril filaments. Many different proteins can aggregate into cross beta sheets, meaning that more than one type of protein is at risk of developing the plaques that cause Alzheimer’s and Parkinson’s
In a published paper of a protein’s crystal structure, how should you interpret dotted lines in the otherwise “solved” protein structure.
The dotted lines indicate intrinsically disordered regions, which due to their dynamic nature don’t have a fixed structure and can’t be seen in crystal structures.
Which has less Gibbs free energy–an unfolded protein or a fully folded protein?`
A fully folded protein
Why is our knowledge of protein sequences greatly outpacing our knowledge of protein structures?
Because it is much easier and cheaper to determine a protein’s primary AA sequence than it is to discern its complete folded structure
What is Alpha Fold? Why are researchers (and pharmaceutical companies) so excited about it? Will it completely replace X-ray crystallography and cryoelectron microscopy? Why or why not?
Alpha Fold is a partially analytical and partially generative AI that outputs a fully folded protein structure when input with the primary amino acid sequence. Its creators won the 2024 Nobel prize in chemistry for it. However, it will likely never fully replace traditional structure determination techniques like x-ray crystallography and cryoelectron microscopy because those two are the gold standards of structure determination. They will probably continue to be used as fact checkers for Alpha Fold’s initial work.
What happens to cellular proteins under conditions of severe heat? Abnormal pH?
They denature (unfold)
What are the various consequences of protein misfolding–either a specific protein misfolding or conditions in which most of your cellular proteins are unfolding.
Main consequence is loss of function
A specific protein not folding correctly impacts the larger system that it was functioning in, which has a range of consequences depending on the unfolded protein’s function and what system it was a part of
In cystic fibrosis, a single protein not folding fast enough leaves it vulnerable to be targeted for degradation before it can reach the plasma membrane, causing the disease
Conditions in which most cellular proteins are unfolding are much more severe, and can easily cause death of the cell(s) and the organism
Misfolded proteins can also cause aggregation/clumping, which can cause diseases like Alzheimer’s, Parkinson’s, liver disease from alpha1-antitrypsin aggregates, etc
What conditions lead to elevated levels of chaperone proteins? Why?
Any kind of stressful conditions will lead to elevated levels of chaperone proteins because all proteins need chaperone assistance to fold properly under stress conditions
Describe the basic cycle of how members of the Hsp70 superfamily (molecular chaperones) mediate protein folding. What are some diverse functions played by members of the hsp70 superfamily within cells? Why might ecologists want to assess Hsp70 levels in their study organism?
Hsp70 follows a basic binding-nonbinding-ATP hydrolysis cycle. They bind to exposed hydrophobic patches, which allows its cycles to slow down folding, allowing the protein to fold correctly
Some Hsp70s can handle proteins as they are being synthesized, some specialize in stress conditions, some delay folding before organelle insertion
Hsp70s often work in teams of many other Hsp70s as proteins exit the ribosome for insertion into the mitochondria (these proteins were once encoded in the mitochondria, now are coded in the nucleus, the Hsp70s guide them back to the mitochondria for function)
If each of the hydrophobic regions of a newly synthesized protein is bound by an hsp70 molecule–why would this keep the newly synthesized protein in a linear (non-folded) configuration?
If each of the hydrophobic regions of an unfolded protein are bound by an Hsp70, then the protein would no longer have any hydrophobic regions exposed to the cell’s aqueous environment, preventing hydrophobic effects and hydrophobic collapse. Since hydrophobic collapse is one of if not the first step in the folding process, the protein will maintain its linear, unfolded configuration
The basic structure of GroEL/Hsp60 chaperonins could be described as a double-chambered “private dressing room” -Explain. What conformational changes occur as ATP binds an is then hydrolyzed and released? What’s the deal with the “changing walls?” How does GroEL/Hsp60 contribute to proper protein folding within the cell?
GroEL/Hsp60 chaperonins are described as dressing rooms because they are shaped like a barrel, formed by two hollow subunits. The unfolded protein enters one of the chambers, attracted to it by its interior hydrophobic regions, and then the chaperonin does ATP hydrolysis to attach the GroES lid and change the walls from hydrophobic to hydrophilic. This forces the protein inside to fold and push its hydrophobic regions inwards. Once its properly rearranged, its released
Can the loss or gain of a couple of non-covalent bonds significantly alter the binding affinity between two proteins? Explain your answer.
Yes. Binding strength is determined partially by the number of noncovalent bonds involved, so the loss or gain of even a few of them will absolutely affect binding affinity.
Protein A and B typically bind together to form a heterodimer. How might elevated temperatures affect the relative levels of [A], [B], and [AB] at equilibrium?
Elevated temperatures make binding more difficult
AB levels would decrease
Protein A and B typically bind together to form a heterodimer. How might a mutation in protein A that decreases its stability (shortens its half-life) and thus lowers the cellular concentration of protein A affect the relative levels of [A], [B], and [AB] at equilibrium?
AB levels would decrease
Protein A and B typically bind together to form a heterodimer. How might increased salt concentrations affect the relative levels of [A], [B], and [AB] at equilibrium?
Salt disrupts ionic and hydrogen bonds (and any other bond with a significant different in electronegativity/dipole moment)
Levels of AB dimers would decrease
Protein A and B typically bind together to form a heterodimer. How might the addition of a “scaffolding” protein to the solution–a protein that can bind to both A and B and has a flexible region in between its A and B binding domains- affect the relative levels of [A], [B], and [AB] at equilibrium?
Increased concentration of AB dimers
KD is the equilibrium dissociation constant. Does stronger binding correspond to a lower or higher KD?
Lower KD
If two proteins A and B bind together with high affinity– which has more energy, the unpaired proteins or the heterodimer?
The unpaired proteins
What is the molecular nature of ubiquitin (Sugar? Protein? Lipid?) Is ubiquitin a large or small molecule in comparison to other macromolecules of its type?
Ubiquitin is a small protein
What is a proteasome?
Proteasomes are three chambered tubes with proteolytic enzymes inside the middle tube. The middle tube breaks down any proteins that enter it. The first and third tubes function as caps that regulate entry into the center tube. Only polyubiquitinated proteins can enter. The end caps also have unfoldase functions to unfold the polyubiquitinated proteins and can recycle and release the ubiquitins.
Depending on the particular case, the E3 ubiquitin ligase alone or in combination with the E2 conjugation enzyme targets the specific substrate and covalently links a ubiquitin either directly to
the target protein or to a ubiquitin that is already covalently attached to the target protein. At what step in the larger process is ATP required?
ATP is required to ubiquitinate E1, the ubiquitin activating enzyme
How does the E3 Ub ligase “know” which proteins to bind?
E3 only binds to proteins with exposed degradation sequences (aka degron motifs or DEAD boxes). If this motif isn’t exposed, E3 won’t bind
In addition to providing a system for destroying misfolded proteins, what else is accomplished by this evolutionarily ancient process of poly-ubiquitination? Under what circumstances might a cell want to destroy an otherwise functional protein?
Polyubiquitination can target any protein for destruction, not just misfolded ones.
A cell would want to destroy an otherwise functional protein during cell cycle developments, differentiation, and protein misfolding.`
When the shape of a protein is altered by either a non-covalent binding interaction or a covalent modification–is the shape change always limited to the physical region of the modification or interaction–or could other regions of the protein be impacted?
Any modification requires a recalculation of the lowest possible energy state, which subsequently forces local refolding and potentially global refolding. This whole process is the conformational change.
What is a GTPase? How does it differ from a kinase or phosphatase? How does it differ from a GTPase activating protein (GAP) or a guanine exchange factor (GEF)?
GTPase hydrolyzes GTP to GDP + Pi. They are noncovalently associated with GTP. Sometimes helper proteins, like GAP and GEF are involved, but these two proteins only remove and replace GDP and GTP. They do not have the hydrolysis activity, GTPase does.
Why are C-G bonds stronger than A-T or A-U bonds?
Because CG pairs are bound with 3 H bonds while AT and AU are bound with only 2
What is meant by the term “DNA melting”? Why does the DNA melting temperature depend on the G-C content?
Melting refers to the unwinding and separation of DNA strands that occurs when the temperature of the DNA molecules is increased enough to allow the hydrogen bonds between the base pairs to be broken while leaving the phosphodiester linkages in the backbone intact. Since GC pairs have an additional H bond, GC rich strands require more heat to melt.
Chromosome definition
A condensed, packaged unit of DNA visible during metaphase
Gene defintion
A distinct sequence of nucleotides, the order of which determines the primary polypeptide sequence of an amino acid
Allele defintion
one of two or more alternative forms of a gene, found on the same spots on the chromosomes. Arises from one or more mutations to the gene.
What’s non-coding DNA? Is most of your DNA coding? What about in yeast?
Non-coding DNA is DNA that is not transcribed into a functional protein or RNA molecule. Only 2% of human DNA is coding, while around 75% of yeast DNA is coding.
Would you expect the nuclei of the interphase cells in your body to all look similar under an electron microscope?
Yes. During interphase, the chromatin is loose in the nucleus and resembles a large plate of spaghetti.
True or false? Deacetylation of histone tails allows nucleosomes to pack together into tighter arrays, which usually reduces gene expression.
True. Acetylation, specifically of lysine residues (which are often found in histone tails) removes a positive charge, which forces the DNA to spread out and the chromatin to subsequently loosen. Deacetlylation therefore does the opposite, it reveals a positive charge which attracts the negatively charged DNA back to it, compressing the DNA and compacting the chromatin, which reduces the transcription machineries ability to access the genes in that region and reduces gene expression.
Regular lysine
Positively charged
Compacted
Monomethylated lysine
Still positively charged (methyl groups are nonpolar)
Compaction
Acetylated lysine
Positve charged is removed
Loose
Definition of the term “histone code”
The patterns of post-translational modifications to the histone tails of the nucleosomes, which determines chromatin compaction and accessibility of the transcription machinery, and therefore gene expression
What is the TBP?
TBP is a subunit of the larger TFIID protein. It is the first protein to bind to the DNA, doing so at the TATA box. It bends the DNA (doesn’t just straddle it!) when it binds and allows the TFIID protein and subsequently the transcription initiation complex to be positioned properly
What happens if a gene has no TATA box?
Then other subunits of TFIID will bind to other promoter elements
Transcription loading process: first through last
TBP/TFIID
TFIIB
TFIIE + TFIIH
RNAP 2 + TFIIF
Would you expect the transcription initiation complex to assemble in vitro–in the absence of DNA that contains a promoter region?
No. The transcription initiation complex is assembled sequentially, with the second and third and so forth steps only occurring after the initial binding to a DNA promoter element (either TATA box and TBP or other). With no promoter region, there would be nothing to bind to, and the assembly wouldn’t happen.
5’mG cap–why can it be thought of as an “upside down,” methylated GTP?
Because the GTP addition to the guanine sugar that provides the methylation is inverted, resulting in a guanine that is upside down in comparison to the rest of the DNA sequence.
Ionic detergents
Have a polar head group and a nonpolar tail
Non-ionic detergents
Detergents that don’t ionize or carry a charge in water
B cells
the type of white blood cell that makes antibodies
Plasma cells
Developed forms of B cells that makes antibodies en masse.
Memory cells
A type of immune cell that remembers a particular antigen
Definition of cytology and immunocytology
Cytology: the examination of a particular cell type
Immunocytology: the examination of a particular type of immune cell
Immunofluorescence and Immunohistochemistry
a lab technique that uses fluorescently tagged antibodies to bind to antigens in a cell for visualization
Why do western blots and other immunocytology experiments use two different antibodies?
The two antibodies provide indirect detection, which increases assay sensitivity and reagent flexibility
At what steps can eukaryotic gene expression be controlled?
Transcription, RNA processing, RNA transport + localization, translation, mRNA degradation, protein activity
How do you choose an appropriate organism or cell line to study?
The organism must do the same process you’re trying to study. Not all organisms do the same things
The same is true for cell lines. Cells specialize. You have to choose a line that has does the process you’re trying to study
Pancreatic cells are good for studying…
Secretory proteins and secretion
Liver cells are good for studying…
Lipids
Red blood cells are good for studying…
The plasma membrane
Muscle cells are good for studying…
Actin and myosin
What facilitates protein binding?
Noncovalent bonds shape the two proteins’ interface. Proteins are bound with hydrogen bonds, hydrophobic interactions, and other noncovalent interactions. The more of these smalle/weak interactions there are, the stronger the bind is
What bonds hold together protein primary structure?
Covalent peptide bonds
What bonds hold together protein secondary structure?
Alpha helices and beta pleated sheets are stabilized by hydrogen bonds in the generic part of the amino acids, not in the R groups
What bonds hold together protein tertiary structure?
Hydrophobic interactions, H-bonds, disulfide bonds, van der walls forces, and some ionic bonds
What bonds hold together protein quaternary structure?
Same as tertiary
Hydrophobic interactions, H-bonds, disulfide bonds, van der waals forces, and some ionic bonds
How are the R groups oriented in an alpha helix?
Outwards from the helix center. About 7 amino acids make up a complete curl
How are the R groups oriented in a beta pleated sheet?
R groups alter directionality every amino acid
This has implications. Barrels or tubes made of beta pleated sheets, esp ones found in membranes, will have their hydrophobic residues face outwards, so that they interact with the lipid membranes. Every R group should alter hydrophobic-hydrophilic
Are protein cores usually hydrophobic or hydrophilic and why?
Oil drop model. In aqueous or other polar environments, the hydrophobic amino acids move to the interior of the polypeptide structure in a motion known as “hydrophobic collapse”
Which steps on the path to a fully folded native state are the fastest?
Formation of secondary structures (alpha helices, beta sheets) and hydrophobic collapse
How does protein diversity arise?
Genetic innovation
Gene duplication
DNA segment shuffling
Explain genetic innovation in the context of protein divesity
An intragenic mutation occurs in the gene, creating a new allele and a new protein
Explain gene duplication in the context of protein diversity
A gene is duplicated, which allows one copy to diverge and create a new protein + allele while retaining fitness from the still functional non-diverged gene
Explain DNA segment shuffling in the context of protein diversity
Segments of DNA coding for a protein or a protein domain are rearranged. This is how domains move to different locations in a protein and how they are added or removed from proteins
For some evolutionarily related proteins, domain structure remains largely unchanged even when the primary sequence has diverged considerably. What implications does this have for BLAST searches?
The whole sequence of the protein may not yield useful results from the BLAST search. Instead, smaller chunks of the sequence must be used
If protein structure is so fundamental to protein function, why do so many proteins have large intrinsically disordered regions?
IDRs let you do more for less. They are unstructured, and can facilitate many different interactions because they can conform to more than one binding domain
One example are histone tails. Because they are made of IDRs, they don’t have a fixes shape, which allows them to be modified by many different types of post-translational modifications
What makes IDRs disordered?
IDRs are made of hydrophilic amino acids. In aqueous environments, those long hydrophilic regions are likely to be floppy because they’re constantly attracted to every passing H2O molecule. The sequences that make up IDRs don’t need to be conserved because as long as the AAs are hydrophilic, it doesn’t really matter what they are or what order they’re in
Do histone tails show up in x-ray crystallography experiments?
No! Histone tails are made of IDRs, which can’t show up in crystallization experiments because they can’t reproduce a singular structure to crystallize with. Their structures are somewhat random and therefore can’t form the necessary lattice crystal pattern
Chaperones
Proteins/molecules that lower the activation barrier between misfolded and native states of a protein. They act catalytically to speed up the folding process. Many proteins routinely enlist chaperones under normal conditions. All proteins enlist chaperones under stressful conditions.
Chaperones can initiate the folding of larger proteins, massage protein clients whose function requires frequent shape shifting, keep proteins linear as they are threaded through membrane channels, and prevent aggregation during stress such as heat shock or abnormal ion balances
Hsp70
Binds to exposed hydrophobic patches
Requires ATP
May require multiple binding cycles, meaning binding-nonbinding-ATP hydrolysis patterns
Cycles slow down folding helping the protein fold correctly
Some handle proteins as they are being synthesized, others specialize in stress conditions or delay folding before organelle insertion
Often work in teams of many other Hsp70s as proteins exit the ribosome for insertion into a mitochondria
Chaperonins
Heat shock proteins, prevent misfolding during high heat based stress
Chaperonin “private dressing rooms”
Double chamber folding machines
Protein enters a chamber, attracted to its hydrophobic walls
GroES, a secondary molecule, caps the chamber and seals the protein inside
GroEL now undergoes ATP hydrolysis, which makes the chamber more confined and changes its interior walls from hydrophobic to hydrophilic
The protein inside is forced to rearrange so that its hydrophobic residues are inwards in its core
GroES is removed and the protein is released
Amyloid plaques
The aggregations that cause Alzheimer’s, made up of cross beta sheets
Lewy bodies
The protein aggregations that cause Parkinson’s disease
What happens to milk in low pH?
The casein proteins aggregate, curdling the milk
What determines binding strength between two proteins?
The shape of the fit/conformation and the number of noncovalent bonds involved in the binding
What is an equilibrium constant?
A measure of binding strength at a particular temperature
K = products / reactants
What deltaG values do biologically relevant interactions have?
-5 to -10 kcal/mol range
What does a deltaG value of -1 tell us about binding affinity between two proteins?
It tells us the proteins basically never bind, no binding affinity
What does a deltaG value of -20 tell us about binding affinity between two proteins?
It tells us the proteins are basically always bound to each other, all the time. 100% binding affinity
Why do siamese cats have dark colored legs, ears, and tails as adults when they have full white coats as kittens?
Siamese cats have a temperature sensitive allele for tyrosinase, the protein that colors their coats dark. This allele only works at low temperatures, so when they are kittens and are always at high temperatures their tyrosinase never works, but as adults it can work in their outer extremities such as their legs, ears, and tails.
What kinds of things are impacted by differences in protein binding affinities?
Allelic differences create genetic variation in a population
The specificity of drugs and potential off target side effects
Protein purification protocols
Determining the appropriate drug concentrations needed to stimulate or inhibit a receptor
Examples of cells using controlled synthesis and destuction to regulate protein function
Cyclin-dependent kinases + cyclin
Proteosome “chambers of doom”
How are proteins marked for destruction in the proteasome?
Different E3 Ubiquitin Ligases recognize different substrates. Fun fact: there’s around 600 different E3s in humans!
There’s a full chain needed ubiquitinate a protein, and this chain must repeat many times for the protein to be polyubiquitinated
First an ubiquitin is added to E1, an Ubiquitin Activating Enzyme. This requires ATP → AMP + Pi + Pi. Then E1 must bind to E2, a Conjugating Enzyme, for the ubiquitin to be transferred to the E2. Then E2 must bind to E3, the Ubiquitin Ligase. Now E3 can search for a degradation sequence (aka degron motif, aka DEAD box) on a target protein. Once found, it binds to the sequence and transfers the ubiquitin from the E2 to an amino group on a lysine side chain of the protein. Now the cycle must repeat again for each subsequent ubiquitin.
Why is cyclin destroyed instead of cyclin+CDK when progressing the cell cycle forwards?
Because it’s more efficient and cost effective
Phosphates on serine, threonine, and tyrosine
Drives the assembly of a protein into larger complexes
Methyl groups on lysine
Condenses chromatin
Acetyl groups on lysine
Loosens/activates chromatin
Palmityl groups on cysteine
Fatty acid addition that drives protein association with membranes
NAG on serine and threonine
Controls enzyme activity and gene expression in glucose homeostasis
Monoubiquitin on lysine
Regulates the transport of membrane proteins in vesicles
Polyubiquitin on lysine
Targets proteins for destruction in the proteosome
A protein has just received a post-translational modification. Now what?
It must change shape. The modification, regardless of what it is, changes the protein and requires a recalculation of the lowest possible energy state. Often, this recalculation results in refolding. Maybe not of the whole protein, but definitely at least part of it. This is the conformational change.
What happens to Hog1 MAPK when cellular salt concentrations are increased?
Hog1 is transiently phosphorylated, which alters its nucleus localization. It now rapidly enters the nucleus, but once inside it is then dephosphorylated and leaks back out into the cytosol. The salt forces the phosphorylation, which pushes Hog1 into the nucleus
Non-covalent and other post-translational modifications are cheaper than destroying and resynthesizing new proteins and are usually reversible, but this doesn’t mean that non-reversible changes aren’t common. What’s an example of a non-reversible modification?
Notch-mediated cell signaling between adjacent cells. Notch ligands from adjacent cells bind to notch receptors, which initiates cleavage of an interior protein, allowing it to travel into the nucleus where it will act as a transcription factor. The cleavage is non-reversible.
Under what conditions would it be advantageous or essential to destroy a protein instead of turning it on/off with reversible post-transcriptional modifications?
Cell cycle progression
Differentiation/specialization
Misfoled protein cleanup
GC vs AT
GC has 3 H bonds, AT has 2
What is chromatin
DNA + proteins
Euchromatin
loose, accessible, unwound, less dense
Heterochromatin
tight, inaccessible, wound, transcriptionally inactive
Why are textbook pictures of chromosomes somewhat misleading?
Most of the time, our chromosomes are in the G1 phase, meaning they’re in spaghetti blob form. They’re only in those neat, compacted chromatid shapes in metaphase. But because it’s pretty easy to chemically arrest cells in metaphase and snap some clear pictures of the sister chromatids, those are the representations most often seen in biology textbooks.
How much of the human haploid genome is transcribed?
Only 55%, 95% of which is intronic
How much of the human genome codes either proteins or functional RNAs?
2-3%
Are telomeres and centromeres usually heterochromatic or euchromatic?
Heterochromatic. Those regions are usually pretty transcriptionally repressed and inactive. They’re gene poor regions, especially for the telomeres, whose explicit function is to not be genes.
What histones are found in normal nucleosomes? Where will you not find these histone variants?
H2A, H2B, H3, H4 make up the core octamer, H1 is the linker histone that caps the loop around the end
You won’t find these guys in the centromeres. Centromeres have their own distinct histone variants, and their own distinct histone modifications. Their histones have changes in the amino acid sequence of both the core histone structures and the histone tails.
Female mammals have two X chromosomes. But this is too many! Only one per cell is needed. How is this conundrum solved?
Barr bodies / X chromosome inactivation
At some point in cell replication, the cell will “choose” one X to inactivate. This “choice” is somewhat random
The inactivated chromosome is completely condensed and creates a Barr body, which is a fun little black dot that is neat to see under a microscope.
Calico cats get their coat colorings from genes on their X chromosome(s), hence why their colorings are random and why true calico cats are always female
HAT
Histone acetyl transferase
Puts acetyl groups on lysine residues, opens local chromosome regions
HDAC
Histone deacetyl transferase
Removes acetyl groups, closes local chromosome regions
Epigenetics
“above genetics”. Patterns of histone modifications are reproduced during DNA replication such that daughter cells have the same patterns of accessible/non-accessible genes. These are heritable patterns of gene expression that aren’t based directly on DNA sequence
Which control mechanisms (for eukaryotic protein expression) are most impactful? Which accounts most for expression variations?
73% is attributed to transcription rates
11% for mRNA degradation
8% for protein degradation
8% for rates of mRNA translation
Regulatory transcription factors (RTFs)
Sequences specific DNA binding proteins
Bind to cis-regulatory elements, aka enhancers
Recognize and bind to specific sequences 5-10 base pairs long
Most include one or more alpha helix motifs to bind to the exposed nitrogenous bases in the DNA major groove
If a mammalian gene doesn’t use a TATA box, what kind of sequence might it use as its promoter instead?
A CpG island
Why do DNA binding proteins often function as dimers?
- Increases specificity. A 6 bp binding sequence randomly occurs 4^6 times. But if binding requires a dimer, then the sequence randomly occurs 4^12 times
- Creates a sharper on/off signal. Binding is synergistic, cooperative. The monomers aren’t found as dimers free in the nucleus, they only become dimers when bound to the binding site. The first monomer to bind usually binds very weakly. The second monomer is the high affinity partner that greatly strengthens the bind.
Types of access DNA promoters and enhancers
Breathing, transcription regulator, and pioneer factors
How does “breathing” allow access to a promoter or enhancer?
A cis-regulatory sequence (enhancer) is on the edge of the nucleosome, and releases/opens from the octamer around 5% of the time.
Binding is occasional
How does a transcription regulator allow access to a promoter or enhancer?
A TR is a molecule that binds to and rips off the sequence from the nucleosome, allowing the RTF to bind
If the cis-regulatory sequence (enhancer) is located near the end of a nucleosome, the TR molecule will bind with 20x LESS affinity than it would to naked DNA
If the cis-regulatory sequence (enhancer) is located near the middle of a nucleosome, it will bind with 200x LESS affinity than it would to naked DNA
How does a pioneer factor allow access to a promoter or enhancer?
Pioneer factors are a class of TR molecules that destabilize the nucleosome, facilitating the binding of a pioneer transcription factor (pTF)
Binding frequency is excellent
How do TFs recognize and bind to specific DNA sequences?
Each binds to a sequence 5-10 bps long
Recognition occurs via the major groove
TFs often work synergistically in pairs/dimers, or in larger complexes
TFs can access less accessible or nucleosome associated cis-regulatory elements (enhancers) through either breathing, TRs, or pioneer factors
How do RTFs influence transcription initiation complexes?
Remember that RTFs have two and sometimes three domains
The RTF binding domain binds to a specific DNA sequence, and the effector domain then recruits a co-regulatory interactor such as an ATP dependent chromatin remodeling complex, a repressor, or an activator
RTF Effector Domain
Topmost domain, always present
Recruits interactors, such as transactivation or repression molecules, other ATP dependent chromatin remodeling complexes
Can fall into repressor or activator categories
Repressors recruit interactors that have HDAC
Activators recruit interactors that have HAT
RTF Flexible Linker Domain
Optional, not all have this. Midsection domain
Usually some kind of dimerization region or regulatory domain, miscellaneous functions including homo and hetero dimerization, nuclear translocation, regulation of various activities
RTF DNA Binding Domain
Bottom domain, always present
Binds to a specific DNA sequence
Mediator, the scaffolding complex
Found in eukaryotes
Huge multiprotein complex that bridges activation domains to the RNAP complex
Interprets the binding state of multiple bound and unbound cis regulators (RTFs) and their coactivators + corepressors
They bind to lots of stuff at the same time, including RNAP 2, interprets and resolved conflicting factors, then communicates its decision to the transcription initiation complex
How do RTFs influence transcription initiation in bacteria?
Bacteria are way more simple
Repressors bind near promoters, which directly prevents RNAP from binding
Activators remove repressors, directly allowing RNAP to bind
Everything happens near the +1 start site
How do RTFs influence transcription initiation from afar in eukaryotes?
Eukaryotic surprise!
Some things can bind way upstream and way downstream
Mediator helps connect the multitude of RTFs and interprets then communicates the resolved signal to RNAP 2
DNA is loose and floppy, plate of spaghetti. Things that are far away can easily influence each other because the DNA isn’t linear
For the most part, transcriptional activators influence gene expression indirectly. They talk to other stuff instead of binding to DNA
What makes cells progressively distinct over time (specialization)?
Specific and differential combinations of transcription factors, including presence and absence of TFs. Combinatorial gene control model. Some TFs can also set off regulatory cascades, indirectly initiating further differentiation.
7 types of transcription regulator activation
Protein synthesis
Ligand binding
Covalent modification
Addition of a 2nd subunit
Unmasking (removal of a 2nd subunit)
Stimulation of nuclear entry (which can sometimes remove a 2nd subunit)
Release from a membrane
Is the entire fully-processed mRNA transcript translated?
No! The 5’ and 3’ UTRs are not translated, nor are the 5’ caps or poly-A tails
What is the 3rd Base Pair Wobble and why should we care?
Multiple codons code for singular amino acids. The differences in the codons for the single AA usually happens in the 3rd letter of the codon, and is usually a pyrimidine (CU) or purine (AG) swap
Important for evolution, mutations are less consequential/disastrous
New research is showing that not all cells express every type of charged tRNA at the same level, which would affect the speed of translation if it weren’t for the wobble substitutions
Accurate translation requires…
The tRNAs to recognize the correct codons
The amino-acyl-tRNA-synthases to load/charge the tRNAs with the correct AA. If these guys make mistakes, the tRNAs will load the wrong AA into the polypeptide
Amino-acyl-tRNA-synthases
There are 20 different types of these synthases. Each one has a specific binding site for one of the 20 AAs
HOWEVER, they can interact with more than one flavor of tRNA
For these suckers to work, they must bind to one of a few specific flavors of tRNA, then to a single specific AA. Only then do they catalyze the high energy covalent ester bond between the tRNA and the AA
ATP driven catalyzation of the high energy covalent ester AA-tRNA bond
Is the mRNA polyA tail encoded in the genome?
No! The tail is attached via poly-A-polymerase during RNA processing before exiting the nucleus. It is NOT encoded in the genome!
What are ribosomes made of?
rRNA and proteins
rRNA
Key to ribosome structure and function
Functions include hybridization (base pairing) sites, and peptidyl transferase activity (AA transfer + peptide bond formation)
4 functions of the protein synthesis team:
Recognition of the start and stop codons to ensure inframe translation
Bringing together appropriately matched mRNAs and loaded tRNAs
Facilitation of peptide bond formation
Maintenance of quality control standards
Ribosomes undergo rounds of assembly and disassembly. Describe.
Small subunit (bottom) binds to the AUG
Large subunit (top) binds
Translation occurs
Termination
Subunits dissociate
repeat!
Which ribosomal subunit forms the peptide bond?
The large one (top) has the ribozyme action that forms the covalent peptide bond
Basics of translational elongation:
Charged tRNA binds to the A site.
Proofreading via eEF-1 in euks and EF-Tu in proks ensures the correct codon-anticodon match
Peptide bond forms via the ribozyme action of the large ribosomal subunit
Translocation via eEF-2 in euks and EF-G in proks reopens the A site and pushes the amino acids + tRNAs to the E and P sites
The tRNA is deacetylated and exits the ribosome
Eukaryotes first elongation factor
eEF-1
Eukaryotes second elongation factor
eEF-2
Prokaryotes first elongation factor
EF-Tu
The induced fit change in the ribosome caused by the tRNA and the small ribosomal subunit acts as a GAP (GTPase Activating Protein)
Prokaryotes second elongation factor
EF-G
eEF-1 and EF-Tu
Well mannered dates, waits for the date to get in the door before leaving
Two-state fidelity timer
There’s a slight pause after the tRNA anticodon binds to the mRNA codon to ensure they actually match
If correct, the GTP attached to these elongation factors is hydrolyzed to GDP + Pi
For EF-Tu specifically, the induced fit change in the ribosome caused by the tRNA and the small ribosomal subunit acts as a GAP (GTPase Activating Protein)
eEF-2 and EF-G
Tokyo subway pushers
Pushes the two tRNAs from the P+A sites to the E+P sites
Reopens the A site
What domains do regulatory transcription factors have? What would be the consequence of mixing and matching those domains to form a hybrid protein?
All RTFs have at least two domains, some have three. All have an Effector domain and a DNA-binding domain, some also have a flexibly linker domain.
Mixing and matching domains would create a new type of RTF. The effector domain recruits other interactor proteins, like repressors (proteins w/ histone deacetylase activity) and activators (proteins with histone acetyltransferase activity). Attaching one kind of effector domain to a different DNA-binding domain would bring whatever activity that effector domain did to a different DNA sequence.
Activator RTFs
Recruit co-activators, which are proteins that have HAT domains
Repressor RTFs
Recruit co-repressors, which are proteins that have HDAC domains
If you compare the sequence of your favorite protein in a human and a chicken–would you expect the nucleotide sequence and the amino acid sequences to be equally similar/divergent?
The nucleotide sequence may differ slightly (because of the third base pair wobble), but the amino acid sequence should remain roughly the same
Process of translation termination:
The release factor is a protein but has the same shape as a tRNA molecule (example of biological mimicry on a molecular level)
The release factor doesn’t do codon-anticodon matching, but it does still recognize the stop codon
It binds in place of a tRNA, ending translation
The release factor also has a GTPase (specifically eRF3-GTP), which hydrolyzes GTP to GDP + Pi when the ribosome complex disassembles
What would happen if a mutation in eEF-Tu (EF-1) accelerated its GTPase activity?
The pause would shorten and the chance of error (wrong tRNA anticodon mRNA codon match) would increase
What would happen if the mutation in a gene for one specific tRNA gene (assume there are multiple gene copies for individual tRNA genes) altered its anticodon such that it was now complementary to one of the three stop codons?
Instead of terminating translation, translation would continue and longer polypeptide chains would be made, loss of original protein function
Dicer
Endonuclease protein that cuts RNA into short segments
Cuts miRNAs and siRNAs into double stranded fragments
Argonauts
Initially binds to a double stranded siRNA or miRNA molecule, but once bound one strand is released leaving only a single stranded guide RNA in place
Single stranded guide RNA + argonaute protein + other misc. proteins = the RNA Induced Silencing Complex (RISC)
Once the RISC is bound to its target mRNA, the argonaut subunit catalyzes the cleavage of the mRNA, which will then be degraded
miRNAs
Micro RNAs
Come from RNA molecules that are transcribed and processed in the nucleus, then are exported to the cytoplasm as double stranded precursor molecules
The double stranded precursors bind to Dicer
Once cut by dicer, it is transferred to an Argonaut protein. One strand of the ds molecule is selected to be released, the other remains bound to Argonaut as the guide strand
Also guide RISC to target mRNAs
Usually only part of the miRNA, known as the seed, base pairs with the target mRNA. This matching is imprecise, and allows miRNAs to target hundreds of endogenous mRNAs
Leads to the target mRNA being degraded or translation inhibited
siRNAs
Small interfering RNAs
Derived from longer double stranded RNA molecules, which are either produced in the cell or delivered experimentally
The double stranded precursors bind to Dicer
Once cut by dicer, it is transferred to an Argonaut protein. One strand of the ds molecule is selected to be released, the other remains bound to Argonaut as the guide strand
Direct RISC to bind to specific mRNAs.
Targeting is precise, determined by base pairing between the siRNA and the target mRNA
