Eukaryotic Transcription 4

Question 1

what is the role of the heptad repeats in the CTD of the largest subunit of RNA pol II?

Answer 1

• Helps bind to DNA and is essential for proper functioning of RNA pol II
• 2 and 5 are Serienes

Question 2

Which large basal transcription factor could you capture with an affinity column comprised of a synthetic CTD (heptad repeats)?

Answer 2

TFIIH

Question 3

Does Mediator bind hyperphosphorylated or hypophosphorylated CTD?

Answer 3

hypophosphorylated

Question 4

Which protein (in which factor) responsible for phosphorylating Ser5 of the CTD? What happens regarding transcription as a consequence?

Answer 4

• the Cdk7 of TFIIH
• transcription is paused

Question 5

What happens when RNA pol II pauses after initiation?

Answer 5

Capping complex, the splicing complex and the add poly(A) complex bind the CTD

Question 6

How is the pause alleviated?

Answer 6

TEFb phosphorylates Ser2 of the CTD and the two negative factors, DSLIP and NELF

Question 7

What removes Ser5 as elongation proceeds?

Answer 7

a phosphatase recruited by TFIIF

Question 8

What keeps TFIIF from recruiting the phosphatase while bound to the promoter in the PIC?

Answer 8

contact with TFIIB

Question 9

TFIIF

Answer 9

• promoter targeting of RNA pol II by binding to TFIIB and reduced nonspecific DNA interactions of pol 2
• involved in initiation and elongation; decreases pausing and protects elongation complex from arrest
• stimulates phosphatase activity which removes phosphates from the CTD - prepares the CTD for the next round of initiation and binding to the mediator complex

Question 10

TFIIF is usually bound to which enzyme?

Answer 10

RNA pol II

Question 11

what is the role of TFIIB Zn-ribbon in recruitment of TFIIF/RNA pol II

Answer 11

in the N-terminus of TFIIB

Question 12

Which basal factor decreases pausing once elongation is under way?

Answer 12

TFIIF

Question 13

Which subunit of TFIIE (alpha or beta) can be traced back to Archaea?

Answer 13

alpha

Question 14

Xerma pigmentosa

Answer 14

mutations in TFIIH (helicase) can result in xerma pigmentosa in humans

Question 15

what are the components of TFIIH?

Answer 15

XPD, XPB, Mat1, cyclin H, cdk7

Question 16

XPD

Answer 16

• helicase in TFIIH
• not active as a helicase during transcription but is required for NER
• can be traced back to arachaea and contain RecA domains

Question 17

XPB

Answer 17

• helicase in TFIIH
• can be traced back to arachaea and contain RecA domains

Question 18

Mat1

Answer 18

• TFIIH
• components of the CAK

Question 19

cyclin H

Answer 19

• regulated by the cdk8 module of mediator
• apart of CAK

Question 20

cdk7

Answer 20

• apart of CAK

Question 21

Which two subunits of TFIIH have domains obtained from the bacterial RecA protein?

Answer 21

the two helicases (XPB and XPD)

Question 22

Explain the ratchet mechanism for helicase action by XPB.

Answer 22

• XPD helicase activity does not participate in transcription but is still needed structurally to properly align with the kinase domain
• XPB helicase lobes 1 and 2 ratchet along the downstream DNA by binding and releasing the backbones of the minor groove

Question 23

Which component of TFIIH independently participates in the regulation of the cell cycle?

Answer 23

CAK

Question 24

Reinitiation Scaffold Complex: which 3 components leave each cycle of transcription?

Answer 24

TFIIB, TFIIF and RNA pol II

Question 25

what acts as the "trigger" that ends the initiation phase and begins elongation?

Answer 25

CTD phosphorylation

Question 26

what does hyperphosphorylated CTD dislodge from?

Answer 26

TBP and mediator

Question 27

when does RNA pol 2 initiate and clear the promoter?

Answer 27

when CTD is hyperphosphorylated

Question 28

what happens to the CTD in absence of TFIIB?

Answer 28

the CTD is dephosphorylated by phosphatase activity stimulated by TFIIF

Question 29

termination of the transcription cycle

Answer 29

- RNA pol II/TFIIF complex leaves the template, forms complex with the mediator, reinitiates with the CTD in the underphosphorylated state

Question 30

what is the CTD?

Answer 30

- carboxy-terminal domain - largest subunit and consists of an array in amino acid repeats (7) - tyr-ser-pro-thr-ser-pro-ser - 42 repeats in humans, 26 in yeast

Question 31

TFIIE

Answer 31

- binds RNA pol 2 and recruits TFIIH - modulates TFIIH helicase, ATPase and kinase activity - assists TFIIH in promoter melting

Question 32

TFIIH

Answer 32

- closely associated with TFIIE binding and regulation - promoter melting through helicase activity - promoter clearance by CTD kinase activity - functions in nucleotide excision repair

Question 33

TFIIH core

Answer 33

- 2 helicases - DNA binding activity 6-7 subunits

Question 34

protein kinase complex

Answer 34

- TFIIK in yeast, CAK everywhere else - 3 subunits - phosphorylates the CTD of RNA pol 2 large subunits and activates cyclin-dependent kinases

Question 35

what are the functions of TFIIE/H in transcription?

Answer 35

- phosphorylates CTD which facilitates promoter clearance - together with TFIIE, metls the transcription start site using 3' - 5' helicase - required for formation of first phosphodiester bond in RNA synthesis

Question 36

Steps in formation of the open promoter complex

Answer 36

1-Promoter DNA is positioned above the cleft (DNA Channel) in the polymerase. 2-Cleft opens and the DNA swings into the cleft. 3-Clamp closes and distorts the DNA in the region just downstream from the TATAA in preparation for melting.

Question 37

what percentage of yeast RNA pol 2 promoters can melt without TFIIH?

Answer 37

18%