Lecture 4

Question 1

What are the GTFs of RNA pol II

Answer 1

TFIIA, TFIIB, TFIID, TFIIE, TFIIH

Question 2

What are the directions that TATA containing and TATA-less promoters undergo for transcription?

Answer 2

Transcription initiates and proceeds in one direction (Uni-directional)

Question 3

What direction of transcription occurs at other promoters (CpG islands)?

Answer 3

Transcription initiates in both directions, but the polymerase stalls and falls off DNA in one of the directions and then proceeds (bidirectional transcription), txn proceeds in one direction only towards the open reading frame (ORF

Question 4

How is RNA pol II loaded onto promoters?

Answer 4

By GTFs

Question 5

Where does transcription pause in unidirectional txn?

Answer 5

After pol II initiates txn it pauses at +50 to +200 region

Question 6

Which have more nucleosomes, CpG island rich promoters or TATA rich promoters

Answer 6

CpG rich DNA contains less nucleosomes and are easier to transcribe

Question 7

What is the meaning of the graph experiment?

Answer 7

At CpG islands equal the number of RNA pol initiate both the sense and antisense direction, sense transcripts pause before elongating further, antisense transcripts pauses at the other end of the CpG island and do not continue

Question 8

What do the graphs for ChIP look like for both bidirectional and unidirectional transcription look like?

Answer 8

Bidirectional: Two peaks
Unidirectional: One peak

Question 9

What are the steps to forming the pre-initiation complex?

Answer 9

1. TFIID associates with the promoter

Question 10

What are the two factors that form the TFIID complex?

Answer 10

TBP and TAFs

Question 11

What are the steps to forming the pre-initiation complex?

Answer 11

1. TFIID binds to the TATA box in the promoter DNA - this bends the dna into a kink and holds the dna in place (TBP)
2. TFIIA and TFIIB attach to the promoter
3. RNA Pol II with the attached CTD (Non-phosphorylation carboxyterminal domain) and TFIIF join the complex - the core PIC
4. TFIIE and TFIIH (the rest is a helicase) and its kinase (TFIIH kinase) attach to the complex
5. TFIIH helicase ATP hydrolysis activity facilitates the opening of the double helix of DNA at the txn start site and pushes dna downstream into the pol- Open PIC
6. TFIIB N-term and Pol II melt dna to form txn bubble
7. Pol II initiates txn in the resulting open complex and continues txn away from promoter
8. The TFIIH kinase phosphorylates the CTD tail of Pol II - Closed PIC

Question 12

When does Pol II pause txn?

Answer 12

Between initiation and elongation

Question 13

What are the three molecules that are involved in pausing transcription?

Answer 13

DSIF - elongation factor, pause RNA pol II
NELF - elongation factor, pause and stabilize RNA pol II
PTEFb - kinase, made out of CDK9/CycT, phosphorylates CTD and NELF

Question 14

What occurs in transcriptional elongation with HIV?

Answer 14

1. Short RNA strand that was made before pausing forms a secondary structure called TAR, TAR holds the pol and has inhibitory effect of Cdk9/CycT
2. HIV1 encodes the protein called TAT. TAT binds to TAR to form a stem/loop structure near 5’ end of HIV transcript, TAR also binds cellular cyclin T
3. When bound to TAR, TAT activates Cdk9/CycT kinase and releases the paused RNA pol II
4. More tat is made and viral genes are expressed

Question 15

What is the key to HIV latency?

Answer 15

HIV is dormant but activates upon stress and when organism is weak, slowly killing T-cells

Question 16

What is the function of HIV TAT?

Answer 16

Sequence specific RNA binding protein which binds to RNA copy of TAR sequence, functions as a anti termination factor, permits Pol II to read through transcriptional block, it activates cycle T-cdk9

Question 17

What is cyclin T?

Answer 17

A part of PTEFb, activates kinase cdk9,