Eukaryotic Transcription 4 Flashcards
what is the role of the heptad repeats in the CTD of the largest subunit of RNA pol II?
- Helps bind to DNA and is essential for proper functioning of RNA pol II
- 2 and 5 are Serienes
Which large basal transcription factor could you capture with an affinity column comprised of a synthetic CTD (heptad repeats)?
TFIIH
Does Mediator bind hyperphosphorylated or hypophosphorylated CTD?
hypophosphorylated
Which protein (in which factor) responsible for phosphorylating Ser5 of the CTD? What happens regarding transcription as a consequence?
- the Cdk7 of TFIIH
- transcription is paused
What happens when RNA pol II pauses after initiation?
Capping complex, the splicing complex and the add poly(A) complex bind the CTD
How is the pause alleviated?
TEFb phosphorylates Ser2 of the CTD and the two negative factors, DSLIP and NELF
What removes Ser5 as elongation proceeds?
a phosphatase recruited by TFIIF
What keeps TFIIF from recruiting the phosphatase while bound to the promoter in the PIC?
contact with TFIIB
TFIIF
- 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
TFIIF is usually bound to which enzyme?
RNA pol II
what is the role of TFIIB Zn-ribbon in recruitment of TFIIF/RNA pol II
in the N-terminus of TFIIB
Which basal factor decreases pausing once elongation is under way?
TFIIF
Which subunit of TFIIE (alpha or beta) can be traced back to Archaea?
alpha
Xerma pigmentosa
mutations in TFIIH (helicase) can result in xerma pigmentosa in humans
what are the components of TFIIH?
XPD, XPB, Mat1, cyclin H, cdk7
XPD
- 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
XPB
- helicase in TFIIH
- can be traced back to arachaea and contain RecA domains
Mat1
- TFIIH
- components of the CAK
cyclin H
- regulated by the cdk8 module of mediator
- apart of CAK
cdk7
- apart of CAK
Which two subunits of TFIIH have domains obtained from the bacterial RecA protein?
the two helicases (XPB and XPD)
Explain the ratchet mechanism for helicase action by XPB.
- 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
Which component of TFIIH independently participates in the regulation of the cell cycle?
CAK
Reinitiation Scaffold Complex: which 3 components leave each cycle of transcription?
TFIIB, TFIIF and RNA pol II
what acts as the “trigger” that ends the initiation phase and begins elongation?
CTD phosphorylation
what does hyperphosphorylated CTD dislodge from?
TBP and mediator
when does RNA pol 2 initiate and clear the promoter?
when CTD is hyperphosphorylated
what happens to the CTD in absence of TFIIB?
the CTD is dephosphorylated by phosphatase activity stimulated by TFIIF
termination of the transcription cycle
- RNA pol II/TFIIF complex leaves the template, forms complex with the mediator, reinitiates with the CTD in the underphosphorylated state
what is the CTD?
- 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
TFIIE
- binds RNA pol 2 and recruits TFIIH
- modulates TFIIH helicase, ATPase and kinase activity
- assists TFIIH in promoter melting
TFIIH
- closely associated with TFIIE binding and regulation
- promoter melting through helicase activity
- promoter clearance by CTD kinase activity
- functions in nucleotide excision repair
TFIIH core
- 2 helicases
- DNA binding activity 6-7 subunits
protein kinase complex
- TFIIK in yeast, CAK everywhere else
- 3 subunits
- phosphorylates the CTD of RNA pol 2 large subunits and activates cyclin-dependent kinases
what are the functions of TFIIE/H in transcription?
- 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
Steps in formation of the open promoter complex
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.
what percentage of yeast RNA pol 2 promoters can melt without TFIIH?
18%
