SOS repair
What is the difference between a lesion, a nick, and a Break?
A lesion is a structural abnormality
Nick is ssDNA break
Break is a dsDNA break
CRISPR is made possible by what kind of DNA repair
What is an operator in transcription regulation?
Its the sequence after the promoter that an enhancer or repressor binds to to regulate the activity of transcription.
What is the purpose of the mediator in Eukaryotic transcription?
it bridges or... Mediates (see what they did there), the interaction between the upstream TF proteins and the RNAP.
essentially acts as a responder to upstream transcription factors, notifying the RNAP to upregulate or repress transcription. (often described as a bridge between the two)
What does CRISPR stand for?
Bonus points:
What is a PAM sequence comprised of?
Clustered regularly interspaced short palindromic Repeats
Bonus:
NGG (n for anything)
In order to activate an SOS response this protein must attach to SSDNA and cleave the operator, which binds to the SOS box and suppresses the expression of this same protein. What enables this protein to be expressed?
Bonus points: what are the names of the proteins I'm talking about
RecA repression is intentionally imperfect and allows for some expression. So that when SSDNA is exposed, it can interact with it and cleave the operator LexA, triggering the SOS response.
What are the 3 components needed for CRISPR gene editing
(Hint: Can be DNA, RNA, or Protein)
tract RNA, crRNA, and cas protein (also donor DNA with desired gene and PAM sequence)
Determine the sense and antisense strand in the image
Antisense is the template strand so it's complementary with the mRNA strand; the other is the coding or sense strand.
Where are each of the RNA polymerases in a Eukaryotic cell located?
Bonus points:
What do each of them produce, and which is the main actor in transcription?
RNAP1: rRNA, located in the nucleolus
RNAP2: mRNA, nucleus
RNAP3: tRNA, nucleus
What is the arrow pushing mechanism for mRNA elongation? show me.
answer sheet
in ecoli what is the order of induction of SOS repair polymerases, and what makes some of them error-prone?
Pol 2:least error prone, has exonuclease activity
pol 4: lacks 3'-5' (proofreading) exonuclease activity
pol 5: lacks 3'-5' (proofreading) exonuclease activity and has very poor fidelity incorperates G opposite T half as often as A.
Pol2 is the first responder (1-2 minutes after damage), then pol 4 (timing seems to be unknown), then pol 5 ~50 minutes later (this is the last resort as even poorly functioning is better than dead)
What is sgRNA and why is it used in CRISPR
Combination of crRNA and tracrRNA
What are the names of the -35 and -10 consensus sequences in E.coli.
Bonus points:
What are the eukaryotic equivalents?
-35 TTGACA
-10 TATAAT (pribnow box)
Eukaryotes
-90 - -70 CAAT box
-40 - -100 GC box
-25 - -30 TATA box
Explain the process of assembling the PIC (pre-initiation complex)
1) TATA binding protein (TBP) attaches to TATA box along with a few TAFs (TATA-associated factors) to form TF2D
2) TF2A and TF2B attach to TF2D ans stabilize the complex
3)TF2F attaches to RNAP and associates with the forming complex
4)TF2E and TF2H associate and complete the PIC
TF2H is a helicase that uses ATP to "melt" the promoter and begin transcription
Name each of the following Eukaryotic equivalents.
RNAP for mRNA, tRNA and rRNA
RNAP Beta and Beta' subunits
Pribnow box
Sigma Factor
GreA and GreB
Bonus points:
What do GreA and GreB do?
RNA pol2, pol3 and pol1
RBP2 and RBP1
TATA box
TF2 proteins ABDFEH
TF2S
Bonus points:
GreA prevents back tracking the mRNA (stops the process from stalling)
GreB helps the already backtracked mRNA by cleaving it to get it to out of the stalled state
Explain the steps of NHEJ
1) Ku70 and Ku80 associate with the ends of the double strand break.
2) Mre11 comes in and trims the ends, and a polymerase comes in and adds bases to these frayed ends (pol mew and pol lambda, it's not in the slides so we don't need to know this)
3)Ku proteins align the ends
4) ligated together by Ligase 4 and Xrcc4, after which they dissociate and the (very mutagenic) repair is complete
You and the incredibly cool and studly Dr. Dennis Fineberg are trying to knock out a gene in e.coli using CRISPR. You have designed your sdRNA to match the DNA of the Gene and made sure to have the segment be opposite a PAM sequence to ensure it would cut. After a series of tests its clear that the gene remains inside the plasmid. Where are you going wrong and what do you need to do to fix it?
To knock out a gene, you need to make 2 cuts on either side of the gene so it will actually be removed. You would need to find a section of DNA downstream of the gene and opposite a PAM sequence, then design your sdDNA to match it.
Label the parts of the RNAP and briefly describe their function.
Bonus points:
what is the name of the orange protein inside the RNAP
answer sheet
You and the incredibly cool and studly Dr. Dennis Fineberg are called in to weigh in your biochemical expertise on a particularly confounding case. The condition of a patient at the nearby hospital is slowly deteriorating, and they can't find out why. Upon investigation, you discover that his body is widely underproducing protein. You follow your incredible instinct and check the ribosomes and see no mutations; they are fully functional and plentiful in the cell. You decide to sequence his genes and see no mutations affecting transcription factors but you do notice that the levels of mRNA in his cell are far below normal levels. Upon investigation, you notice that the RNAP in the cell are stalled and failing to make mRNA. What is the most likely cause of his ailment, given the information? Why do you believe that?
It is likely Alpha-amanitin poisoning. The polypeptide enters the funnel and interacts with the bridge helix and trigger loop, preventing NTPs from entering and forming mRNA.
DON'T EAT RANDOM MUSHROOMS!
What were the major findings of the Jinek paper? What made it significant?
In broad strokes, it demonstrated the parts of CRISPR necessary for its function and also showed that combining the crRNA and tracrRNA into sgRNA would still allow CRISPR cleavage to occur. enabling a much easier to design precursor to be made for gene editing.
3 in 1: HR
Before beginning let me explain the rules for this question:
Explain the steps of HR for the following
1. SS lesions
2. SS nicks
3. DS breaks (Eukaryotes)
Lesions
1) Replication fork collapses at the lesion
2) The 2 growing strands move back and BP, forming the Chicken foot (branch migration). RecA works on the strand w/o lesion to regress it and even out the strands
3) Pol 1 extends the shorter strand (the one which BP with lesion strand)
4) Reverse branch migration, the strands resume as expected
SS Nicks
1) DNA production continues until nick where the fork collapses and the strands separate
2) RecA and RecBCD mediate strand invasion to give the shortened DNA a template, creating a holliday junction
3)RuvAB does a branch migration with the other strand
4) RuvC resolves the junction
Double-strand Breaks
1) Rad51 (RecA equivalent) creates 2 Holliday Junctions via moving one of the ss ends to the opposite strand
2) The other break associates with the other strand
3) DNA polymerization and ligation
4) The Holliday junctions are resolved via GEN1 (RuvC equivalent)
You are working with a mutant version of the Cas9 protein. This mutant has a mutation affecting the HNH-like domain. What effect would this have on the function of the Cas9 protein, and would it still be able to perform gene editing?
The HNH-like domain is responsible for cleaving the backbone of the Target strand (the strand that BPs with the crRNA or sgRNA, respectively). A mutation in this region would likely lead to Cas9 protein only cutting one of the two strands, making it unable to perform gene editing.
You are a scientist determining the effects of mutagenic substances on a new strain of E.Coli. Upon exposure to a new mutagen, you see in this E.coli impossibly long strands of mRNA sprouting out of the plasmid. What are 3 possible reasons that could lead to these extended mRNA chains? What will likely happen to this new strain? Will it be viable?
1) Mutation in the intrinsic terminators, the self-complementary strands have been mutated and the hairpins that would form aren't, leading to no strand termination.
2) Mutation in the Rho factor leading to an inability to function and bump the RNAP off the strand. Again, causing no strand termination
3) Mutation in the rut site, preventing the rho factor from associating and again stopping the mRNAs from terminating.
The proteins formed would be misshapen and nonfunctional. Also the energy used to make these mRNAs would be unsustainable. The cell would die very quickly.
You have a eukaryotic mutation leading to under-expression of CTD kinase. What effect does that have on the cell?
Bonus Question:
You have a eukaryotic mutation leading to under-expression of CTD phosphatase. What effect does that have on the cell?
Bonus Bonus question:
is the phosphorylation and dephosphorylation of the CTD energy neutral?
Lack of CTD kinase means that RNAP 2 will have trouble initiating transcription, as it only occurs when RNAP 2 is phosphorylated.
Lack of CTD phosphorylase means that RNAP 2 will have trouble elongating the mRNA, as it only elongates when its unphosphorylated.\
No its not energy neutral
Explain all the figures shown and what conclusions can be drawn.