Section 3 - Genome Control and Gene Expression Flashcards
What is the central question of genome control?
How do I select particular DNA for gene expression?
What is the challenge with transmission of information (DNA -> RNA -> protein)?
Want it to be reliable, but don’t want to mess up DNA (wear)
What is the differences between different cells (in terms of gene expression)?
Different cells read different genes (different euchromatin) to get different protein products
True or false: the genome is organized cohesively
False: it has a disruptive organization
What is the organization of the genome?
Small bits of coding DNA are interspersed with large sections of noncoding DNA
True or false: proteins that work together are usually coded together
False: proteins coded together typically have dissimilar functions
What is the difference between mammallian genomes and bacterial genomes (based on gene expression)?
Similar bacterial proteins can all be transcribed under one promoter, while similar mammalian proteins are all over the genome
When can RNA be the final product (in terms of the central dogma)?
RNA can act as enzymes (gain catalytic activity by folding)
What is transcription?
DNA -> RNA
What step is DNA -> RNA?
Transcription
What is translation?
RNA -> protein
What step is RNA -> protein?
Translation
What does RNA formation rate depend on?
Cell regulation, promotors, etc.
What does the RNA formation rate (partially) dictate?
The amount of protein produced
What is the most common way to control protein production?
Through RNA
Why is RNA the most common way to control protein production?
It is transient and degradable
True or false: all RNA makes the same amount of protein
False: there are various controls that regulate the amount of protein
True or false: if there is higher production of a protein A (compared to another protein B), then protein A is more important
False: there could be other reasons (stability, toxicity, efficiency, etc.) for why A is produced more than B
What describes different levels of proteins in the cell?
The controls in place (tune processes to get result) at every stage
What is the first step in making a protein?
Transcription (DNA is transcribed into RNA)
What is RNA?
A linear polymer made of four different nucleotide subunits
What is the difference between DNA and RNA?
RNA has uracil (DNA has thymine), and RNA has ribose (DNA has deoxyribose)
What is the difference between ribose and deoxyribose?
Ribose has an OH group on the 2’ carbon, while deoxyribose has an H
What base is uracil most similar to?
Thymine
True or false: RNA is always single stranded
False: it can form interesting 3D structures by folding in on itself
How can RNA become more stable?
It can bind to itself to form 3D shapes, such as double helices
True or false: in RNA, the bases must be on the same side of the backbone
False: there is free rotation, so the bases can rotate freely
What is the first step of transcription?
To unwind and open DNA
How many strands act as a template for the RNA?
One of the two strands
True or false: all the template strands are one side of the DNA
False: they can be on either side
What determines which RNA base will bind to the complementary DNA base?
Binding kinetics
How stable are the bonds between RNA and DNA in transcription?
Fairly weak (RNA transcript is easily removed from DNA)
What does RNA polymerase do?
Transcribes DNA into RNA
What enzyme makes RNA from DNA?
RNA polymerase
True or false: RNA polymerase can also stabilize the unwound DNA
True: it can stabilize it while making the RNA transcript
What provides the energy for RNA transcription?
The breaking of the phosphate bonds from triphosphate nucleotides
What are the raw materials to create the RNA transcript?
Triphosphate nucleotides
What enzyme(s) is/are RNA polymerase analogous to?
Helicase (unzip and stabilize DNA) and DNA polymerase (make a nucleotide transcript)
How does RNA polymerase stabilize unwound DNA?
By having the different strands in different channels
Where are the nucleotides found during transcription?
In the ribonucleoside triphosphate uptake channel in RNA polymerase
What does the ribonucleoside triphosphate uptake channel do?
Place where the triphosphate nucleotides are found during transcription
What is meant by a “short loop of association” (in transcription)?
RNA and DNA are bound for a couple of base pairs before RNA dissociates from the DNA
True or false: multiple polymerases can act at one time
True: usually in bacterial cells, multiple polymerases can transcribe a gene at the same time
How come multiple polymerases can act on a gene at the same time?
RNA is almost immediately removed from the DNA
Looking at bacterial transcription, how can you tell what is the start / end of the gene?
By the lengths of the RNA transcript (shorter transcripts means closer to the start of the gene)
Why does DNA look “darker” in the regions with RNA transcripts?
The RNA polymerases are sitting on the DNA
What are some differences between RNA polymerase and DNA polymerase
- RNA polymerase works with RNA nucleotides
- RNA polymerase does not need a primer
- RNA polymerase makes a mistake every 10^4 nt (as opposed to 10^7)
- Dissimilar mechanisms
Why does RNA polymerase not need a primer?
Because the RNA transcript is transient
True or false: RNA polymerase requires a primer to work
False: it does not need a primer
Why does RNA polymerase have a lower accuracy than DNA polymerase?
Because RNA is transient, so it can be easily degraded
Which has the higher accuracy: RNA polymerase or DNA polymerase?
DNA polymerase
What error correcting does RNA polymerase have?
It cannot move forward when the backbone is distorted
What error correcting does RNA polymerase not have?
It cannot remove bases (exonuclease) that are bound to the RNA like DNA polymerase can for DNA
What is mRNA and what does it do?
Messenger RNA codes for proteins
What is tRNA and what does it do?
Transfer RNA act as adaptors between amino acids and mRNA
What is rRNA and what does it do?
Ribosomal RNA catalyze protein synthesis in the ribosomes
What is snRNA and what does it do?
Small nuclear RNA aid in nuclear processes (splicing of pre-mRNA)
What is snoRNA and what does it do?
Small nucleolar RNA process and chemically modify rRNA
What is miRNA and what does it do?
Micro RNA regulate gene expression by blocking translation of mRNAs
What is siRNA and what does it do?
Small interfering RNA turn off gene expression by signaling degradation of mRNA
What is piRNA and what does it do?
Piwi-interacting RNA bind to piwi proteins to protect the germ line from transposable elements
What is lncRNA and what does it do?
Long noncoding RNA usually serve as scaffolds to regulate cell processes (X-chromosome inactivation)
How many RNA polymerases are in bacterial cells?
1
How many RNA polymerases are in eukaryotic cells?
3
What does RNA polymerase I do?
Transcribed 3 rRNA genes (5.8S, 18S, and 28S)
What does RNA polymerase II do?
All protein-encoding genes, plus other smaller RNAS (siRNA, miRNA, most snRNA, etc.)
What does RNA polymerase III do?
tRNA, 5S rRNA, and other small RNAs
What talking about transcription, which RNA polymerase is most likely being mentioned and why?
RNA polymerase II, since we usually talk about making proteins
What does the “S” value refer to in rRNAs?
The sediment value (how heavy it is after centrifugation)
What is a promoter?
The location where the gene starts and RNA polymerase needs to start transcribing DNA
What is needed to position RNA polymerase correctly at the promoter?
General transcription factors (GTFs)
What does GTF stand for?
General transcription factors
What do GTFs do?
Position RNA polymerase correctly, pull apart DNA strands, and release RNA polymerase from promoter
What is the promoter analogous to?
The origin of replication
True or false: an origin of replication is also a promoter
False: they both refer to different processes (DNA replication vs RNA transcription)
Where do GTFs bind on the DNA?
TATA box
What is a TATA box?
A region of DNA rich in A’s and T’s
What is the significance of the TATA box?
Weaker hydrogen bonding, thus easier to pull apart
What does binding of GTFs do to the DNA?
Distorts the backbone, allowing for other machinery to get into place
What is the combination of proteins and GTFs for transcription called?
Transcription initiation complex
Where is the TATA box located?
~25 nt upstream from the gene of interest
What is special about TF2H?
It is a growth factor that includes helicase
What transcription factor includes helicase?
TF2H
True or false: growth factors are relatively small proteins
False: they can be composed of many subunits
What is the significance of the tail region of RNA polymerase?
When phosphorylated, it switches sides and clamps the polymerase onto the DNA
What is the controller of RNA polymerase activity?
The C-terminal tail domain
When do the transcription factors fall off the DNA?
When the RNA polymerase tail is phosphorylated
What transcription factor stays on the DNA after the RNA polymerase tail is phosphorylated?
TF2D
Why does TF2D remain on the DNA, even after the RNA polymerase tail is phosphorylated?
It can reassemble the transcription initiation complex if needed
What makes using unpurified DNA more difficult to use?
More proteins are needed to transcribe DNA
What proteins are needed to transcribe unpurified DNA?
Transcription activators, mediators, and chromatin / histone remodeling complexes
Why are transcription activators needed to transcribe unpurified DNA?
Activator binds far away from the gene to help initiate transcription
Why are mediators needed to transcribe unpurified DNA?
Mediators couple the activators with transcription initiation complex
Why are chromatin / histone remodeling complexes needed to transcribe unpurified DNA?
Chromatin / histone remodeling complexes may be needed to help access the DNA
What is the structure of a mediator?
Fits like a glove for all the protein machinery
Why is meant by purified DNA?
Remove many parts of the transcription initiation complex
When would purified DNA be used?
When you are just trying to transcribe a gene
When would unpurified DNA be used?
When you are studying transcription specifically
What signals does a mediator respond to?
Whether more activators or inhibitors are signaling for the transcription of that gene
What does an activator bind to?
An enhancer region
What binds to an enhancer region?
An activator protein
How large is the (unpurified) transcription initiation complex?
Over 100 proteins
What is the order of assembly of the transcription initiation complex?
Somewhat randomly
What happens after RNA is initially transcribed?
It is modified through covalent modifications and RNA splicing
What covalent modifications are done on RNA to process it?
A 5’ cap and a poly A tail
What is RNA splicing?
Removal of introns (and keeping exons) from the RNA transcript
What are introns?
Noncoding (intervening) regions of RNA that need to be removed
What are exons?
Coding (expressed) regions of RNA that remain in the transcript
What is the purpose of the covalent modifications of RNA?
Stabilization, and determine whether the entire transcript is intact or not
What is a primary RNA transcript?
Unprocessed RNA (no modifications)
True or false: prokaryotic RNA has a 5’ cap
False: it has 3 phosphate groups, but not a 5’ cap
True or false: prokaryotic RNA has a poly A tail
False: it has no covalent modifications to the 3’ end
True or false: after RNA modifications, the entire RNA transcript codes for a protein
False: there are also untranslated regions (noncoding) before and after the coding region
What is the structure of a 5’ cap?
A triphosphate bridge, with 7-methylguanosine
Besides starting RNA transcription, what does phosphorylation of the RNA polymerase tail do?
Recruit specific processing proteins
What happens to processing proteins once they are on the RNA polymerase tail?
They “hop” to the RNA to process it
What is the order of processing proteins on RNA?
Capping factors, then splicing proteins, then 3’ end processing proteins
What determines which processing proteins will bind to the RNA polymerase tail?
How it is phosphorylated (number and place)
True or false: RNA modification happens in real time
True: the modification proteins hop from the RNA polymerase tail to the RNA transcript to process it in real time
What are the 3 steps for RNA capping?
- A phosphatase removes a phosphate group from the 5’ end of mRNA
- A guanyl transferase adds GMP to the mRNA end
- A methyl transferase adds a methyl group
How does RNA splicing occur?
Through transesterifications, which breaks the phosphodiester bonds in two locations
What structures are formed during RNA splicing?
Intron lariats
What is a lariat?
A lasso-like structure that is made up of spliced introns
What is found in every intron?
An activated A
What does an activated A do?
Allows for the formation of the lariat
True or false: all exons and introns are the same size
False: they are all different sizes, which leads to a messy system
What are some reasons that introns may be useful?
Evolutionary, help modify genes, or help make cell specific proteins of similar genes
What is needed for specific splicing of RNA introns?
A specific RNA sequence needs to be recognized
Where are the specific RNA sequences for splicing found?
At the borders of the introns and exons, and around the activated A
What does the spliceosome do?
Induces splicing of RNA
What is the spliceosome?
A protein-nucleic acid complex that splices RNA
What forms the spliceosome?
snRNAs and 7 other proteins
What do the snRNAs do in the spliceosome?
Locate the specific regions of the RNA for splicing
What is the general process of splicing?
- Recognize sequences of RNA
- Pull together to form lariat
- Join ends together to remove lariat
How does an RNA recognize a particular sequence?
Through complementary binding (associations)
What associations are found in a functioning spliceosome?
Protein / protein, and snRNA to transcript RNA
True or false: exon sizes are somewhat uniform
True: within reason, exon sizes are fairly uniform
True or false: intron sizes are somewhat uniform
False: introns can have many different sizes
What does CstF stand for?
Cleavage stimulation factor
What does CPSF stand for?
Cleavage and polyadenylation specificity factor
What does CstF do?
Splices mRNA from RNA polymerase and helps end the reaction
What does CPSF do?
Helps other proteins bind (PAP and poly-A-binding proteins)
What does PAP stand for?
Poly-A polymerase
What does PAP do?
Adds ~200 adenines to the transcript
What enzyme adds the poly-A tail?
PAP (poly-A polymerase)
True or false: PAP needs a template to work
False: it just adds A’s
What do poly-A binding proteins do?
Bind to the poly-A tail to stabilize it (prevent looping) and keep it linear
What proteins bind to the poly-A tail to stabilize it?
Poly-A binding proteins
What are poly-A binding proteins analogous to?
Single strand DNA binding proteins
True or false: all RNA molecules leave the nucleus
False: many RNAs do not leave the nucleus and are degraded
What RNAs do not leave the nucleus?
Introns, improperly formed RNAs (no 5’ cap and poly A tail), RNAs that were unnecessarily transcribed (changed conditions), RNAs that stay in the nucleus (spliceosome) and RNAs that have an siRNA attached
What happens if an RNA does not meet the criteria to leave the nucleus?
They encounter the nuclear exosome, which contains RNA exonucleases to cleave the RNA
What are nuclear pores?
Tightly regulated channels that control when enters and leaves the nucleus
What is more tightly regulated: plasma membrane or nuclear membrane?
The nuclear membrane
What does a complete RNA transcript have?
Multiple proteins to signal that it is properly made and can leave through the nuclear pores
What does the nuclear export receptor do?
Allows the nuclear pore to open and let the RNA transcript / attached proteins into the cytosol
What protein opens the nuclear pore for the completed RNA transcript?
The nuclear export receptor
When does the nuclear export receptor fall off the RNA transcript?
After the nuclear pore is open and the RNA transcript is in the cytosol
What does CBC stand for?
Cap-binding complex
What does the CBC do?
Helps protect the 5’ cap
What protein helps protect the 5’ cap?
The CBC
What does EJC stand for?
Exon junction complex
What does the EJC do?
Denotes where two exons have come together
What protein denotes where two exons have come together?
The EJC
What does SR proteins stand for?
Serine / arginine proteins
What do SR proteins do?
Their function is not known currently
What does hnRNP stand for?
Heterogeneous nucleus ribonuclear proteins
What do hnRNPs do?
Help with binding to the ribosome
What proteins help with binding to the ribosome?
hnRNPs
Where do protein initiation factors bind to?
The 5’ cap (CBC)
What is the structure of RNA ready for translation?
A loop (due to poly A tail)
When does the CBC come off of the RNA?
When it forms a loop (signaling it is ready for translation)
What happens if the RNA is not in a loop structure in the cytosol?
It gets degraded by enzymes
What happens if the RNA is in a loop structure in the cytosol?
It is ready to be translated into a protein
Why does an RNA not in a loop structure get degraded by enzymes?
Enzymes recognize the free poly-A tail, signaling the RNA for degradation
True or false: rRNAs are final gene products
True: they do not encode for proteins, and they are themselves functional
How many rRNAs are there?
4
True or false: rRNA is chemically modified
True: it is chemically modified
How is rRNA chemically modified?
Through methylation and isomerization of uridine (uracil + ribose)
How many rRNAs make up the large subunit?
3 (5.8S, 5S, and 28S)
How many rRNAs make up the small subunit?
1 (18S)
Where is the nucleolus found?
Within the nucleus (not a physical barrier)
What does the nucleolus do?
Contains the rRNA machinery (and perhaps other protein-nucleic acid complexes such as telomerase) (localization)
What does the nucleolus contain?
rRNA genes, precursor rRNA, mature rRNA, rRNA processing enzymes, and ribosomal protein (localization)
When do the large and small subunits of a ribosome combine?
When attached to mRNA
How is mRNA decoded?
In sets of 3 (codon)
How long is a codon?
3 nt
What is the genetic code?
The rules to translate a codon into an amino acid
True or false: there are no exceptions to the genetic code
False: there are some exceptions, such as in the mitochondria
True or false: the genetic code is redundant
True: some amino acids have multiple codons that lead to the same amino acid
What codon is the start codon?
AUG (Methionine)
True or false: every protein starts with methionine
False: while it is the start codon, it is usually cleaved off
Where is the redundancy (usually) in a codon sequence?
The third nucleotide
What is a reading frame?
Where the RNA sequence is first read
What is the significance of the reading frame?
Important to align amino acids, since it can drastically change the protein
True or false: most frame shifts will not encode usuable proteins
True: frame shifts dramatically alter the amino acid sequence
What is the adaptor between mRNA and the amino acids?
tRNA
What is the structure of tRNA?
Cloverleaf shape, ~80 nt long
What are the critical regions of tRNA?
Anticodon (links with codon), and amino acid binding site
What is a wobble position?
The third site of the anticodon, which has weak hydrogen bonding
What is the significance of the wobble position?
Helps explain redundancy (3rd position is less important than the first two)
True or false: the wobble is the same between eukaryotes and prokaryotes
False: there are different wobbles between the two types of cells
True or false: every codon is represented by a unique tRNA
False: due to wobble, not all tRNAs are needed
True or false: tRNAs are processed similarly to mRNAs
True: they are both spliced and chemically modified
What enzyme attaches amino acids to tRNAs?
Aminoacyl-tRNA synthetases
What does aminoacyl-tRNA synthetase do?
Attaches an amino acid to a tRNA
In most cells, how many aminoacyl-tRNA synthetases are there?
20 (one for each amino acid)
How does an aminoacyl-tRNA synthetase get the correct amino acid?
By having a high binding affinity for only that amino acid (size / interactions)
How does an aminoacyl-tRNA synthetase get the correct tRNA?
Through binding with its anticodon
How does the editing site of aminoacyl-tRNA synthetase work?
Only closely related amino acids can go into the editing site, where it is hydrolyzed
What directions are proteins made in?
From N-terminal to C-terminal
Where does the energy come from to form a peptide bond?
The high energy linkage between the tRNA and the amino acid
What does the ribosome do?
Site for protein synthesis
What is the ribosome composed of?
~50 proteins and 4 rRNAs
What does the large subunit of a ribosome do?
Catalyzes the peptide bond
What does the small subunit of a ribosome do?
Framework to hold mRNA and site for tRNA binding
How many sites are on a ribosome?
4 (1 for mRNA, 3 for tRNA)
What are the three sites on a ribosome that tRNA binds to?
E (exit), P (peptidyl), and A (aminoacyl)
Which sites hold the tRNA tight?
A and P sites
What are the 4 steps of protein synthesis?
- tRNA binding
- Peptide bond formation
- Large subunit translocation
- Small subunit translocation
What happens during tRNA binding?
The correct tRNA enters the A site (proper codon-anticodon binding)
What happens during peptide bond formation?
The carboxyl group is released in the P site, and attached to the amino group in the A site
What enzyme is active during peptide bond formation?
Peptidyl transferase
What does peptidyl transferase do?
Creates a new peptide bond during protein synthesis
What happens during large subunit translocation?
The large subunit shifts relative to tRNA and mRNA (P -> E, A -> P)
What happens during small subunit translocation?
The small subunit shifts three nucleotides
What happens at the A site of the ribosome?
A new amino acid is added to the growing chain (tRNA enters)
What site does a new tRNA (amino acid) enter and get added to the growing chain?
A site
What happens at the P site of the ribosome?
Holds the growing amino acid chain
What site holds the growing amino acid chain?
P site
What happens at the E site of the ribosome?
The tRNA leaves the ribosome
What site does the tRNA leave?
E site
Where is peptidyl transferase found?
Part of the ribosome
Why is a tRNA always found in either the P or the A site?
Holds everything (mRNA, subunits of ribosome) together
True or false: during protein synthesis, there is always a tRNA in either the P or the A site
True: this ensures that the ribosome is together
What do elongation factors do?
Drive protein synthesis in the forward direction (kinetics), provide energy, and partially provide a proofreading mechanism
What reactions do elongation factors help in?
- Binding of correct tRNA to the mRNA
2. Preventing large subunit from going backwards on mRNA
What is a ribozyme?
An enzyme that functions due to RNA
What is an example of a ribozyme?
The ribosome
Why would enzymes require RNA to function?
To locate other nucleic acids through that RNA
What does rRNA do in the ribosome?
Positions tRNA / mRNA, and forms peptide bonds
What is peptidyl transferase composed of?
Solely rRNA
What tRNA is needed to start translation?
Initiator tRNA
What does initiator tRNA bind to?
Start codon (AUG)
True or false: initiator tRNA binds to only the small subunit
True: it is the only tRNA that can do this (bind without the large subunit)
What does the small subunit do once it is bound to an initiator tRNA?
Finds an mRNA (with initiation factors) and scans for the first AUG
When does the large subunit bind to the small subunit?
Once the first AUG is found on the mRNA
True or false: the initiation factors fall off of the mRNA once protein synthesis begins
False: they can stay on the mRNA so it can be translated again
What is the control of protein synthesis?
When the initiation factors fall off of the mRNA, it cannot code for more proteins
What codons cause translation to end?
UAA, UAG, or UGA
True or false: there are tRNAs associated with the stop codons
False: there is a release factor instead
What does the release factor do?
Binds to stop codon, causing the end of translation
What does peptidyl transferase do when a release factor is present?
Adds a water to the end of the protein to break the chain
What step of translation does the ribosome fall apart?
Large subunit translocation
Why does the ribosome fall apart during large subunit translocation (at the end of translation)?
No tRNAs are bound in the A or P sites to hold everything together when the large subunit moves
What is release factor made out of?
Protein
What is the structure of a release factor?
Similar to tRNA
What are polysomes (or polyribosomes)?
Combinations of ribosomes, proteins, and mRNA
What is the purpose of a polyribosome?
Allows for multiple proteins (from the same mRNA) to be formed at once
Where does most of the free energy of the cell go towards?
Protein synthesis
What bonds (and how many) are split to make a peptide bond?
4 high-energy phosphate bonds (two to put amino acid on tRNA, two to move ribosome and form peptide bond)
True or false: protein synthesis is energetically costly
True: a lot of energy is needed to create a protein
What happens if an mRNA is not spliced properly?
It will likely reach an aberrant stop codon (found in intron)
True or false: EJCs can only tell the boundary between two exons
False: they can also be used to determine if the mRNA is spliced properly
If an mRNA is not spliced properly, what can you say about the stop codons and the EJCs?
There is at least one ECJ downstream of a stop codon (which shouldn’t normally happen)
How does a ribosome know if an mRNA is not spliced properly?
If there is an ECJ downstream of a stop codon (which shouldn’t normally happen)
What do UPFs do?
Trigger mRNA for degradation if it isn’t spliced properly
True or false: proteins are functional right after translation
False: they need to be chemically modified to be useful
What does nascent polypeptide mean?
Unfolded protein
How can a protein be chemically modified?
Phosphorylation, co-factors, etc.
What do molecular chaperones do?
Help fold proteins by stabilizing intermediate structures
What proteins help with protein folding?
Molecular chaperone proteins
What are some examples of molecular chaperone proteins?
Heat shock proteins
True or false: molecular chaperone proteins can only hook onto the polypeptide chain
False: they can also hook onto necessary chemical modifications
Without molecular chaperone proteins, what dictates protein folding?
Thermodynamics
True or false: the polypeptide starts folding after being released from the ribosome
False: it starts folding in real time (while it is being produced)
What are “on-folding” and “off-folding” pathways?
Pathways that determine the proper and improper ways a protein can fold
What do molecular chaperone proteins do (in terms of the folding pathways)?
Move a protein from an “off-folding” pathway to an “on-folding” pathway
What happens if protein misfolding is irrecorverable?
The chaperone proteins take the polypeptide to the proteasome for degradation
Besides the flow of information, what does the steps of the central dogma suggest?
Each step can be controlled from DNA -> RNA -> protein
True or false: cells have different genomes since they have different functions
False: cells all have the same genome
How can cells have the same genome but different functions?
They can selectively control which genes are expressed, and thus which proteins are expressed
How is gene expression a spatial problem?
Need to locate the proper genes on the chromosome
How is gene expression a temporal problem?
Need to respond to external stimuli through gene expression
What were the (general) experiments to show that cells contained the whole genome, and not different pieces?
Information from a differentiated cell could be used to produce a new organism
What were some species used in the experiments to show that cells contained the whole genome?
Frogs, carrots, and cows
True or false: mRNA expression for the same gene in different cells is the same
False: there are subtle differences
True or false: a subtle change in mRNA expression leads to a subtle change in protein expression
False: since protein synthesis is highly controlled, there can be a large difference in protein expression based on a subtle difference in mRNA expression
What are the 6 steps to control gene expression?
- Transcriptional control
- RNA processing control
- RNA transport and localization control
- Translation control
- mRNA degradation control
- Protein activity control
True or false: external cues can drive gene expression
True: these stimuli can change gene expression
True or false: cells respond to the same stimuli the same way
False: based on the signaling determinants, there can be different responses to the same stimuli
Why is it common to use one cell type in a lab?
Cells have varying responses, so it is easiest to study one cell response in depth
What are gene regulatory proteins?
Proteins that interact directly with DNA to turn genes on or off
What are some examples of gene regulatory proteins?
Lambda repressor and lac repressor
Where do gene regulatory proteins usually bind?
The major groove on DNA
Why do gene regulatory proteins bind to the major groove on DNA?
Can read the sequence based on the noncovalent interactions
What interactions can be read from the major groove?
- H atom
- H-bond acceptor
- H-bond donor
- Methyl group
How come gene regulatory proteins do not bind to the minor groove?
There is no specificity in the binding chemistry (an A-T looks the same as a T-A)
What is the importance of the major groove?
Can use the structures found in the major groove to read the DNA sequence
What is the difference between the major groove and the minor groove?
Can see more of the chemical substance in the major groove
What leads to the specificity of the major groove in reading the DNA sequence?
The specific pattern of interactions is unique for each base pair
What is used to read the DNA sequence (structurally)?
The major groove
What are the recognition sites for gene regulatory proteins?
Short DNA sequences
True or false: each recognition site is recognized by a specific protein
True: this increases the specificity of gene expression
How many different recognition sites are there?
Over 1000
True or false: there are strong interactions between the DNA and gene regulatory proteins
False: it is a weak, reversible interaction
How many interactions are there between gene regulatory proteins and DNA?
Around 20
What are the mechanisms that gene regulatory proteins can bind to DNA?
Helix-turn-helix, zinc finger, beta sheets, and leucine zippers
What is the helix-turn-helix motif?
Two alpha helices with a bend inbetween them
How does the helix-turn-helix motif bind to DNA?
One helix is the DNA recognition helix, and the other one binds to the backbone to stabilize it
True or false: the helix-turn-helix motif binds as symmetric dimers
True: two monomers are needed to bind to either side of the DNA
What is the consequence of needed a symmetric dimer for the helix-turn-helix motif?
The DNA recognition site must be palindromic
How far apart are the alpha helices in the helix-turn-helix motif?
3.4 nm
Why are the alpha helices in the helix-turn-helix motif 3.4 nm apart?
This is the size of one turn of DNA
What is a zinc finger?
A motif that uses zinc to hold an alpha helix and beta sheet for DNA binding
True or false: one protein corresponds to one zinc finger
False: one protein can have many zinc fingers for recognition
What tradeoffs must be considered when considering how many zinc fingers are in a protein?
Specificity vs. ease of regulation
In a zinc finger, what does the zinc atom bind to?
Four specific amino acids in the protein
What determines the sequence a zinc finger will recognize?
The amino acid structures of the zinc finger
How do beta sheets work in DNA recognition?
Two beta sheets can bind together to form a DNA recognition site (similar to helix-turn-helix)
What do all the mechanisms of DNA recognition have in common?
They use protruding amino acids
What are protruding amino acids?
Amino acids that stick out of the protein
What do protruding amino acids do?
Recognize the DNA sequence through noncovalent interactions
True or false: a protruding amino acid has a single contact with the DNA base pair
False: it can have either a single contact or multiple contacts
What is a leucine zipper?
Two alpha helices that dimerize due to protruding hydrophobic amino acids (such as leucine)
True or false: the leucine zipper binds as symmetric dimers
False: they can bind as either homodimers or heterodimers
What is the consequence of leucine zippers being either homodimers or heterodimers?
They can recognize either a symmetric sequence or an asymmetric sequence
True or false: it is easy to predict the binding between gene regulatory proteins and DNA
False: there are complicated interactions that are hard to predict
What does the tryptophan repressor do?
Regulates the production of tryptophan in bacteria
What does the operator do?
Binding site of repressors to regulate gene expression
What is the operator?
A DNA segment that is the binding site for a regulator
Where is the operator found?
Within the promoter (or far away, depending on the site)
When is the tryptophan operon active?
When there is no repressor (and thus no tryptophan in the cell) bound to the operator
When is the tryptophan operon inactive?
Where the repressor (bound to tryptophan) is bound to the operator
Why is the tryptophan repressor used in labs?
It is extensively studied and easy to control
What happens when a repressor is bound to the operator?
RNA polymerase cannot bind to the promoter, and thus cannot transcribe the gene
What is a positive regulator (in terms of gene expression)?
A protein that increases the likelihood for transcript formation when bound
How do positive regulators work (in terms of gene expression)?
Aid in RNA polymerase binding, or activating a bound polymerase
What are the possible combinations of regulatory protein actions?
- Repressors are activated by addition of a ligand
- Repressors are activated by removal of a ligand
- Activators are activated by addition of a ligand
- Activators are activated by removal of a ligand
If a gene is controlled by a repressor activated by addition of a ligand, when is the gene transcribed?
When there is no ligand present, so the repressor is not bound to the operator
If a gene is controlled by a repressor activated by removal of a ligand, when is the gene transcribed?
When there is ligand present, so the repressor is not bound to the operator
If a gene is controlled by an activator activated by addition of a ligand, when is the gene transcribed?
When there is ligand present, so the activator is bound to the operator
If a gene is controlled by an activator activated by removal of a ligand, when is the gene transcribed?
Where there is no ligand present, so the activator is bound to the operator
What is unique about the lambda repressor?
It can activate certain genes when bound, and can inhibit other genes when bound (can do both functions)
What determines whether the lambda repressor will activate or inhibit a gene when activated?
The position of the operator relative to the promoter
What controls the lac operon?
Levels of glucose and lactose
True or false: controls only function at one site
False: they can be coupled to multiple sites
What is an example of an operon that have controls coupled at multiple sites?
The lac operon
When is the lac operon gene transcribed?
When there is low amount of glucose, and high amount of lactose
What regulators mediate the lac operon?
A lac repressor, and a glucose activator
When is the lac repressor active?
When there is lactose present
When is the glucose activator active?
Where there is glucose absent
Why is the lac operon used in labs?
It is well understood and easy to control
In a lab, how can the lac operon be used and controlled?
By having specific media, a gene of interest can be activated or inhibited
True or false: operators only work at very short distances
False: they can also work at very long distances
How can operators regulate genes when they are very far away?
Through DNA looping (loop back to the gene of interest)
What is an enhancer?
A DNA sequence where an activator or a repressor can bind to regulate gene expression
What does an enhancer do?
Allow for binding of activators or repressors to regulate gene expression
How does a regulatory protein ensure it binds to two enhancer sites?
When bound to one site, it has a large affinity for the other site as well
How does DNA looping work?
A protein binds to two regions in the DNA, which causes it to loop
In terms of general transcription factors, how do eukaryotic cells differ from prokaryotic cells?
Eukaryotic cells need 5 general transcription factors, while prokaryotic cells need 1 transcription factor
In terms of genome arrangement, how do eukaryotic cells differ from prokaryotic cells?
Prokaryotic cells have genes arranged in operons, while eukaryotic cells do not (regulate each gene individually)
What is the significance of eukaryotic cells not having operons?
Each gene needs to be regulated individually (as opposed to a group of genes regulated together)
In terms of regulatory proteins, how do eukaryotic cells differ from prokaryotic cells?
Eukaryotic cells have 100s of regulatory proteins, while prokaryotic cells are controlled by a few
In terms of DNA storage, how do eukaryotic cells differ from prokaryotic cells?
Eukaryotic cells have DNA arranged into chromatin, while prokaryotic cells do not
What is the significance of eukaryotic cells packaging their DNA into chromatin (in terms of gene expression)?
Allows for more opportunities to control gene expression
What are promoters?
DNA sequences where general transcription factors bind
What are regulatory sequences?
DNA sequences where regulatory proteins bind
What does a mediator do?
Mediates the interactions between the regulatory proteins and the polymerase
What is the structure of a mediator?
Shaped to associate nicely with the regulators (activators and repressors)
Besides enhancing the transcription of a gene, what do enhancers do?
Modulate the chromatin sequence to allow for transcription
How is DNA (in terms of its gene sequences) wrapped into the histones?
The TATA box is within the wrapping, and the regulatory domains are outside of the wrapping
What is the significance of the TATA box being wrapped within the histones?
Not accessible for gene transcription when wrapped
What is the significance of the regulatory domains being wrapped outside of the histones?
Accessible for regulatory proteins to come and regulate the wrapping of DNA (and allow for subsequent transcription)
What are the possible results of a regulator binding to DNA wrapped around a histone?
Remodeling, removal, replacement, or modification
What is synergy?
Multiple activators have a more than additive effect on gene transcription
True or false: gene expression is an additive process
False: it is a synergistic process
How is gene expression a synergistic process?
While the activators themselves may lead to 1 UT (unit of transcription), together they may lead to 100 UT, not 2 UT
Which regulatory proteins are most important to gene expression?
The regulatory proteins close to the gene / promoter
How do regulatory proteins far away from the gene affect the mediator (in terms of gene expression)?
May help synergistically, but are most likely not required for transcription to occur
What are the possible mechanisms of gene repression?
Competitive DNA binding, direct interactions with the general transcription factors, masking the activation site, and recruitment of chromatin remodeling complexes
How does competitive DNA binding work in repression?
Overlapping sequences lead to a repressor binding, and not an activator
How does direct interactions with the general transcription factors work in repression?
Repressor binds to the transcription factor, preventing the looping of DNA that would allow the activator to bind
How does masking the activation site work in repression?
Repressor binds to the activator, preventing the activator from binding to the mediator
How does recruitment of a chromatin remodeling complex work in repression?
The chromatin remodeling complex can wrap up DNA so it is inaccessible by the translation machinery
What is an insulator?
A DNA sequence that blocks an enhancer from working on the wrong gene
What is the purpose of a barrier sequence?
Prevents interaction with heterochromatin
What is the purpose of insulators?
Make sure that enhancers far from the gene of interest work on that gene, and not other, closer genes
What is post transcriptional control?
mRNA can be modulated after it is transcribed
What is a riboswitch?
A small RNA segment that binds to metabolites
What does a riboswitch do?
Turns an RNA polymerase on or off based on its change in shape
How does a riboswitch change shape?
Upon binding to a ligand (ex: guanine)
What is alternative splicing?
Having different exons to encode different proteins
What is the consequence of alternative splicing?
Can have the same gene coding for different proteins
What regulates alternative splicing?
Activators and repressors
True or false: alternative splicing involves the removal of introns and exons
False: it is only concerned with removing certain exons (not introns)
What are the (eventual) products of alternative splicing?
Cell specific proteins (different “versions”)
True or false: alternative splicing can change the properties of the protein
True: through altering the coding mRNA, the protein can be different
How can alternative splicing alter the properties of a protein?
By changing the amino acid chain, and consequently the noncovalent interactions, in the protein
If a protein is going to be membrane bound, what types of amino acids will it need?
Hydrophobic
If a protein is going to be secreted, what types of amino acids will it need?
Hydrophilic
What is RNA editing?
Transcribed RNA can be edited by adding various nucleotides
How does RNA editing occur?
Through guide RNAs
How do guide RNAs work in RNA editing?
Guide RNAs find specific sequences, rip apart the mRNA, and add nucleotides
What increases the stability of mRNA?
The 5’ cap and the poly-A tail
What enzyme degrades the poly-A tail?
Deadenylase
What does deadenylase do?
Break down the poly-A tail to degrade mRNA
What does RNAi stand for?
RNA interference
What is RNAi?
Using small RNA strands to bind to mRNA and prevent translation
What RNAs are used in RNAi?
siRNA
