RR4: Proteins required to initiate eukaryotic transcription Flashcards
What is runoff assay?
It’s a way to find out which proteins are involved in transcription and how effective a promoter is or the rate of transcription of certain genes.
It’s a DNA template that’s driven by the adenovirus promoter (example)
You put the promoter upstream of a segment in a plasmid.
Cut the plasmid to have a linear plasmid.
We add the DNA template and nuclear extract, the proteins involved in transcription will assemble on the promoter.
The RNA polymerase would make its way through the DNA segment and fall right off when it reached the end.
It makes RNA that corresponds to the specific segment. Then, we can quantify the amount of RNA made.
After a runoff assay, what are techniques to quantify the amount of RNA made?
Using probes that are reverse complementary to that region.
Add radioactive ribonucleotide to the reaction, so that the RNA molecules are charged by radioactivity, so it creates radioactive products.
Then, by acrylamide gel electrophoresis, followed by autoradiography.
What are the roles of RNA pol 2?
Transcription of mRNA - snRNA - siRNA - miRNA.
Encodes proteins.
RNA splicing.
Chromatin-mediated repression.
Translation control.
Why would we modify RNA pol 2?
To change and modify gene expression.
Why would we want to know which proteins work with RNA pol 2?
To ensure that those proteins are going to the genome and activate the expression of a given gene.
How can we know what’s necessary for transcription to happen?
First, we purify the proteins involved in RNA transcription by nuclear extract. In vitro, when we combine the factors that are associated with RNA pol2, we realize that transcription happens. We try with different factors to see which proteins are essential for transcription.
What are the transcription factors necessary for transcription that we can find in the nuclear extract?
TFIIA
TFIIB
TFIID
TFIIE
TFIIF
TFIIH
Which transcription factor interacts with the TATA box?
TFIID
Which subunit that makes up TFIID binds the TATA box?
TBD
What does TBP do?
TBP: TATA-box Binding Protein
Binds the TATA box tightly and interacts with the TATA box via a minor groove interaction. The CTD of TBP binds to the minor groove and distorts the double helix.
TBP maintains the double helix in that formation.
It also increases the efficiency of transcription with RNA pol1, 2 and 3.
What is CTD?
C-terminal domain.
In TBP and it’s only found in RNA pol 2.
Is TBP essential for promoters that don’t have a TATA box?
Yes. TBP will interact with other regions of the promoter.
Is TBP required for efficient Pol 1, 2 and 3 transcription?
Yes, all 3 require TBP.
What brings TFIID to the promoter?
The TATA-box or other promoter will recruit TFIID.
Why would we need to create a kink in the double helix to initiate transcription?
It creates open regions in the double helix, so it’s easier to access it.
What are the subunits forming TFIID?
TAFs (1-13) : TBP Associated Factors
TBP
What is the role of the TBP Associated Factors?
TAFs interact with other elements that contribute to transcription. That’s why TBP can also work with promoters that don’t have a TATA box.
What are the steps to initiate transcription with the transcription factors?
- TATA box recruits TFIID
- TBP binds to the TATA box on the promoter
- The TAFs interact with other regions of the promoter
- TFIIB and TFIIA are recruited on the promoter to create the upstream promoter complex (TFIID, TFIIA, TFIIB)
- RNA pol 2 and TFIIF are recruited to form a Core PIC
- TFIIE, TFIIH and TFIIH kinase are recruited to form a Closed PIC
- ATP creates an Open PIC
What does Open PIC mean?
Pre-Initiation Complex.
It creates a transcription bubble to initiate transcription. It needs to be open to have a single-stranded template DNA in the transcription bubble.
Why do we need ATP to go from a Closed PIC to an Open PIC?
Because TFIIH has ATP-dependant enzymatic activities. So, to melt the DNA and create an open PIC, ATP is required.
What makes the transition between initiation of transcription and elongation?
When the PIC is open, if you provide the complex with RNTP, polymerase 2 will take off. That’s the switch from initiation to elongation.
What is in TFIIH?
900L of packed HeLa cells
XPB (NER)
XPD (NER)
p44 (associated with DNA repair)
p8 (associated with DNA repair)
TTDA (associated with DNA repair)
cdk7 (cell cycle)
MAT1 (cell cycle)
cyclinH (cell cycle)
Very packed
What are XPB and XPD?
XPB and XPD are DNA helicases found in TFIIH that are involved in nucleotide excision repair system.
What is Xeroderma pigmentosum?
Disease caused when the patient has a faulty nucleotide excision repair system (NER).
Those people can’t go in the sun.
