RR4: Proteins required to initiate eukaryotic transcription

Question 1

What is runoff assay?

Answer 1

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.

Question 2

After a runoff assay, what are techniques to quantify the amount of RNA made?

Answer 2

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.

Question 3

What are the roles of RNA pol 2?

Answer 3

Transcription of mRNA - snRNA - siRNA - miRNA.
Encodes proteins.
RNA splicing.
Chromatin-mediated repression.
Translation control.

Question 4

Why would we modify RNA pol 2?

Answer 4

To change and modify gene expression.

Question 5

Why would we want to know which proteins work with RNA pol 2?

Answer 5

To ensure that those proteins are going to the genome and activate the expression of a given gene.

Question 6

How can we know what’s necessary for transcription to happen?

Answer 6

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.

Question 7

What are the transcription factors necessary for transcription that we can find in the nuclear extract?

Answer 7

TFIIA
TFIIB
TFIID
TFIIE
TFIIF
TFIIH

Question 8

Which transcription factor interacts with the TATA box?

Answer 8

TFIID

Question 9

Which subunit that makes up TFIID binds the TATA box?

Answer 9

TBD

Question 10

What does TBP do?

Answer 10

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.

Question 11

What is CTD?

Answer 11

C-terminal domain.
In TBP and it’s only found in RNA pol 2.

Question 12

Is TBP essential for promoters that don’t have a TATA box?

Answer 12

Yes. TBP will interact with other regions of the promoter.

Question 13

Is TBP required for efficient Pol 1, 2 and 3 transcription?

Answer 13

Yes, all 3 require TBP.

Question 14

What brings TFIID to the promoter?

Answer 14

The TATA-box or other promoter will recruit TFIID.

Question 15

Why would we need to create a kink in the double helix to initiate transcription?

Answer 15

It creates open regions in the double helix, so it’s easier to access it.

Question 16

What are the subunits forming TFIID?

Answer 16

TAFs (1-13) : TBP Associated Factors
TBP

Question 17

What is the role of the TBP Associated Factors?

Answer 17

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.

Question 18

What are the steps to initiate transcription with the transcription factors?

Answer 18

1. TATA box recruits TFIID
2. TBP binds to the TATA box on the promoter
3. The TAFs interact with other regions of the promoter
4. TFIIB and TFIIA are recruited on the promoter to create the upstream promoter complex (TFIID, TFIIA, TFIIB)
5. RNA pol 2 and TFIIF are recruited to form a Core PIC
6. TFIIE, TFIIH and TFIIH kinase are recruited to form a Closed PIC
7. ATP creates an Open PIC

Question 19

What does Open PIC mean?

Answer 19

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.

Question 20

Why do we need ATP to go from a Closed PIC to an Open PIC?

Answer 20

Because TFIIH has ATP-dependant enzymatic activities. So, to melt the DNA and create an open PIC, ATP is required.

Question 21

What makes the transition between initiation of transcription and elongation?

Answer 21

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.

Question 22

What is in TFIIH?

Answer 22

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

Question 23

What are XPB and XPD?

Answer 23

XPB and XPD are DNA helicases found in TFIIH that are involved in nucleotide excision repair system.

Question 24

What is Xeroderma pigmentosum?

Answer 24

Disease caused when the patient has a faulty nucleotide excision repair system (NER).
Those people can’t go in the sun.

Question 25

Which kinases are required for the activation by phosphorylation of very important cell cycles, kinase-dependant?

Answer 25

cdk7, MAT1 and cyclinH

Question 26

What would be all the roles of TFIIH?

Answer 26

Cell cycle
DNA repair
Transcription
Melting DNA
Involved in the phosphorylation of the C-terminal domain (CTD) during initiation

Question 27

Which sections of the genome are more actively repaired?

Answer 27

The sections that are heavily transcribed are repaired more effectively.
Regions around the start site are easily repaired.

Question 28

What are the steps of the formation of a Pre-Initiation Complex (PIC)? (to initiate transcription)

Answer 28

1. eukaryotic promoter (tata box)
2. tata box recruits TFIID
3. Binding of TBD to the TATA box and binding of other TAFs (TBD associated factors) to elements around the start site. The CTD of TBD binds to the minor groove and create a kink in the double helix.
4. Binding of TFIIA and TFIIB
5. Binding of TFIIF and RNA pol 2 (core pic)
6. Binding of TFIIE and TFIIH
7. Stable closed PIC
8. Add ATP and NTPs to being elongation by RNA pol 2

Question 29

We know that cdk7 is involved in the phosphorylation of important cell cycles with MAT1 and cyclinH, but what is the other role of cdk7?

Answer 29

Once TFIIH is recruited by the pre-initiation complex, the cdk7 of TFIIH phosphorylates the CTD of RNA pol2.

Question 30

What’s the role of TFIIH in the phosphorylation of chromosomes during transcription?

Answer 30

When the CTD is phosphorylated, it’s during active transcription.
When TFIIH is recruited by the pre-initation complex, the core pic becomes a closed pic. The kinase cdk7 of TFIIH phosphorylase the CTD of RNA pol 2. ATP is required to switch from closed to open PIC and then transcription starts.

Question 31

What are the minimum requirements for giving rise to transcription?

Answer 31

General transcription factors (TF elements) (proteins)
RNA pol 2
DNA strand with a promoter