RR4

Question 1

What method can be used to see/isolate/examine transcription factors?

Answer 1

Liquid chromatography!
This is good because we want to be able to mix it back together after.

Question 2

How does liquid chromatography work?

Answer 2

Nuclear extract is separated into columns and there are combinations for specific fractions that allow transcription in RNA Pol II. This method allows seperation of extracts which identifies important transcription factors.

Question 3

What are the function of the transcription factors: TF2A, TF2B, TF2H, and TF2D?

Answer 3

They work together to create RNA product.

Question 4

Why is the transcription factor TF2D important?

Answer 4

It is very important because it interacts with the TATA box and the TBP subunit.

Question 5

What is TBP?

Answer 5

It is a TATA box binding protein. It binds to minor groove and kinks DNA and distorts it (saddle appearance). It is critical in TATA box binding and in vitro (no TATA). Class 1 and class 3 base transcription.

Question 6

is TBP in TF2D?

Answer 6

Yes it is inside. All subunits in TF2D are “TBP associated factors.” These are referred to as RAF.

Question 7

Why is there still a need for TATA-less transcription?

Answer 7

There are some TAF’s that interact with other elements around the start site (downstream and upstream). It is fine without a TATA box.

Question 8

Describe the process of pre-initiation? (RNA Pol II transcription)

Answer 8

1) TF2D binds to promoter (may or may not have TATA) and it interacts tightly with DNA, summons TF2A and TF2B
2) TF2A, TF2D, TF2B form the upstream promoter complex and they stabilize the complex.
3) Now that it is stabilized, RNA pol II can react with the complex, and bind to TF2F, which binds to the upstream promoter complex.
4) “core pre initiation commplex” - gene promoter can now activate.
5) TF2E and TF2H comes into complex to stabilize it.
6) With ATP and dATP, DNA will open, RNA Pol II can now interact. And now elongation begins. (need RNTPs)

Question 9

What is TF2H?

Answer 9

It is an important transcription factor. It contains 2 DNA helicases and ‘ATP dependent activity,”
It will melt DNA which opens up the transcription bubble.
It has around 9 different polypeptides;
XPB + XPD needed for ner
P44: DNA repair
P8: DNA repair (last protein on gel)
CDK7 + mat I: cell cycle control

• contains helicase involved in Xeroderma pigmetosum. Faculty NER cells can’t repair skin. Some genome lesions repair faster than other, TF2H is higher in some regions. This is why TF2H is coupled with transcription couple repair.

Question 10

Which polypeptide is responsible for 1st phosphorylation?

Answer 10

CDK7 is.
It activates drive of the cell cycle.