Exam 3- L13 Flashcards
How many different eukaryotic RNAP are there? Which one are we focusing on?
There are three types of eukaryotic DNA (I, II, and III).
We will be focusing on RNAP II, which is responsible for pre-mRNA synthesis.
What are the three different DNAP responsible for?
RNAP I -> pre-rRNA
RNAP II -> pre-mRNA
RNAP III -> tRNA, spliceosomal
How could you distinguish between different polymerase activity?
You could add in a specific toxin and see which polymerase is affected. They all have different effects to the toxin at different concentrations.
What is the largest subunit of a RNAP in euk?
The CTD tail.
What is the CTD tail?
The CTD tail is the subunit of RNAP that has multiple copies of the same, specific sequence. (YSPTSPS).
These tails help recruit protein factors needed for splicing, capping, or terminating.
5 of these 7 residues bear -OH groups, therefore they are hydrophilic and phosphorylatable.
Why is phosphorylation of the CTD tail important?
Phosphorylation of the CTD tail is important for transcription and RNA processing.
What kind of RNAP with its CTD tail can initiate transcription?
An RNAP with an unphosphorylated CTD tail.
What are transcription factors?
They act like sigma factors in prokaryotes. They make a platform for RNAP to come and sit on the promoter sequence.
What do general transcription factors do? (3)
- Attract RNAP to the promoter site
- Determines start point and direction of transcription.
- Basal Level Transcription
What are Gene-Specific Transcription Factors?
They are used for transcriptional control.
Polymerization of RNA is the SAME between prokaryotes and eukaryotes, but in eukaryotes, what do you need?
Helper proteins (transcriptional factors).
What are elongation factors used for?
Both RNAP in prokaryotes and eukaryotes, elongation factors are proteins that move along with the RNAP and are associated with EDITING via nuclease activity to deal with incorrectly placed bases.
What two ways can the RNAP edit incorrectly synthesized RNA NT?
- It can stall just like a DNAP does when a base in incorrectly positioned and that stalling will allow the polymerase to reverse the reaction (if it can quickly). If this were to happen, that means the pyrophosphate must still be present within the active site.
- As it is elongating, it can sometimes stall and the RNA will fall out of the polymerase and there are endonucleases that will cleave the RNA.
What transcription factor is involved in nucleotide excision repair?
TFIIH is involved in nucleotide excision repair, along with 5’ -> 3’ and 3’-> 5’ helicase activity.
It also has three subunits (Cdk7/Mol5, Cyclin H, and MAT 1) that makes up a kinase complex that phosphorylates Ser-5 of the CTD during transcription initiation.
What are two types of promoters that you can have?
- Focused/Sharp-Type Promoter (very specific, only one possibility)
- Dispersed/Broad-Type Promoter (not specific, more than on possibility)
Genes that are expressed at a low rate have what kind of features?
Many genes that are transcribed at a low rate DO NOT contain a TATA box or initiator.
Instead, they contain C-G rich regions or CpG Islands of 20-50 NT upstream from the start site.
Transcription of these genes can begin at any one of these multiple possible sites over an extended region (Dispersed Promoter).
What kind of features would you see with a sharp-type promoter?
Some of the main elements you will see with a sharp-type promoter is a TATA box (-31 to -36). It has a certain sequence just like a promoter sequence in prokaryotes.
You will also have a BRE region (-37 to -32) that has a specific sequence recognized by proteins and transcription factors (i.e. TFIIB)
Along with those two mean features, you can also have an INR (initiator) or DPE (downstream core promoter element) that allow binding of transcription factors that will help establish the pre-initiation complex.
What is one difference for the process of transcription when comparing additional proteins other than RNAP for eukaryotes and prokaryotes?
In prokaryotes, the sigma-factor trucks along with the RNAP.
In eukaryotes, proteins and transcription factors are going to FIRST bind and define the promoter sequence and then recruit the polymerase to the location.
What is the first transcription factor that is recruited to the promoter sequence? Second?
The first TF recruited to the site is the TBP (TATA box binding protein). Which is a saddle like structure that sites right on top of the DNA molecule.
TBP will then recruit TAF (TATA box association factors). These two proteins make up what is called the TFIID.
TFIIA and also come in when TAF does, it just stabilizes the complex
What is the mechanism of TBP binding to DNA?
The TBP uses beta sheets to interact with DNA and it distorts the MINOR GROOVE, so that it can come into contact with the minor groove.
In doing so, it bends DNA so that it is not in its normal alpha-helical structure, flattening it.
TBP also has aromatic AA that will integrate between the bases allowing it to stabilize DNA.
What is the next protein to be recruited after TBIID has been established? What is its significance?
TFIIB come in and bind to the BRE site of the promoter.
TFIIB is important in dictating the orientation/direction of the promoter site. (it has the sigma-3 loop, similar in prokaryotes).
What happens after TFIID and TFIIB have bound?
TFIID and TFIIB established that platform for RNAP.
Now, the polymerase must be recruited to the pre-initiator complex.
TFIIF is going to associate with RNAP and bring it to the site.
After that, TFIIE and TFIIH are recruited to the site.
What kind of addition proteins are recruited to the site besides TF? What is their function?
In addition to the general TF, the polymerase also needs to about with a mediator complex.
It is a set of proteins that interacts very specifically with the RNAP and the TF to induce a conformation change of RNAP so that transcription can begin.
Since incoming NT are in front of the RNAP, the RNAP will need to be associated with nucleosome remodeling and histone modification complexes.
So recap all all of the TF and their function.
- TFIID
- TBP -> binds to the TATA box and distorts DNA
- TAF -> recognizes downstream areas - TFIIB -> Binds to the BRE region, dictates direction of polymerase, has that ‘sigma-3 loop’
- TFIIA -> binds to TAF and stabilizes the PIC
- RNAP + TFIIF -> recruits RNAP to the PIC
- TFIIE -> Helps with TFIIH activity
- TFIIH > has helicase and phosphorylation activity.
What is the role of TFIIE?
It is a protein needed for the activity of TFIIH.
What is the role of TFIIH?
It is a helicase that opens up DNA to create an open complex in both the 5’ -> 3’ and 3’ -> 5’ direction. Just like all helicases, it needs ATP.
TFIIH also phosphorylates the CTD tail on the RNAP to recruit RNA processing proteins.
What happens as soon as TFIIH opens up DNA?
You will have abortive transcription. But once it starts to go, TFIIH will phosphorylate the CTD tail, specifically serine-5 to recruit capping enzymes.
This phosphorylation will cause RNAP to stop because when serine-5 was phosphorylated, it also recruited negative elongation factors.
Negative elongation factors stop the RNAP from continuing synthesis, until that cap is added onto the 5’ end of the mRNA.
What happens after the cap has been added?
Another set of phosphorylation will occur at the CTD tail. It will phosphorylate the serine-2, which will produce the elongating polymerase along with recruit spliceosomal machinery that possess exonuclease activity.
The RNAP can elongate after this phosphorylation because the phosphorylation of serine-2 causes the dissociation of the negative elongation factors.
Overview of the phosphorylations of serines.
First, the CTD tail must be unphosphorylated to allow RNAP to bind to the PIC.
- TFIIH phosphorylates Serine-5, which will recruit capping enzymes along with negative elongation factors.
- Then you will get another phosphorylation at Serine-2, which will also phosphorylate the negative elongation factors to initiate elongation. The spliceosomal machinery is also recruited during this phosphorylation.
Effects of phosphorylation: the simple version.
Serine -5 -> recruits capping enzymes and negative elongation factors (induces promoter clearance)
Serine -2 -> turns off negative elongation factors and recruits spicing machinery (induces elongation)
What specific thing phosphorylates the serine 5?
CDK7 of TFIIH phosphorylates serine 5.
What specific thing phosphorylates the serine 2?
CDK9 of P-TEFb
What removes the phosphate from serine-5? what is the effect?
A phosphatase will remove the phosphate from serine-5 on the CTD tail, which will initiate polyadenylation/termination.
