Eukaryotic Transcription 2 & 3 Flashcards
TBP
- a component of the positioning factor that is required for each type of RNA polymerase to bind its promoter
- mass of 800 kD
what is the TBP for RNA polymerase 2?
- TF2D
- consists of TBP and 11-14 TAFs (TBP associated factors
how does TBP bind to DNA?
- TBP binds to the TATA box in the minor groove of the DNA
- forms a saddle around the DNA and bends it 80 degrees
- 30 kD
what determines the polarity of transcription?
- TBP
- based on the asymmetry in the TATAA sequence itself and interactions with TF2B
how does TBP insert a kin in DNA?
inserts 2 phenylalanines
where does TF2B bind?
- binds to C- terminal stirrup
- plays a critical role in determining the start site for transcription in association with TBP
B-linker helix
- apart of TF2B
- melts the promoter 20 bp upstream from TATAA
B-reader helix
- apart of TFIIB
- contacts -8 of the initiator element
what does the B-reader helix and B-reader loop do?
- contact the 5 end of the nascent transcript
B-reader loop
contacts position -1
the binding of what protein is the first step in initiation?
- binding of TFIID
- other transcription factors bind to the complex in a defined order - extends the length of the protected region on DNA
- when RNA polymerase II binds to the complex, it initiates transcription
how is TBP delivered to the promoter?
by TFIID
TAFs 1 and 7
bind InR and other downstream elements
TAFs 11 and 13
block c-stirrup of TBP
TAF 1
blocks DNA binding cleft and n-stirrup of TBP
what is the process of loading TBP onto DNA?
- TAF1/7 in lobe C bind to downstream elements (InR)
- this positions TBP over the DNA 3- bp upstream, which allows the DNA to compete off the binding of TAND1 and TAF1
- TBP scans the DNA for tightest binding (no TATA sequence)
- TAF4 recruits TFIIA which displaces TAND2 and TAF1 binding to the stirrup pf TBP
- TBP fully engages the DNA to introduce the 80 degree bend, which displaces TAF11
- release of TBP from lobe A, this unlocks the C stirrup of TBP
- the zinc-ribbon of TFIIB recruits TFIIF (RAP40) which is attached to RNA pol 2
- TFIIF interactions displace TAF4 and lobe C interactions with the InR. TFIID falls off but remains nearby due to interactions with upstream activators
where is each lobe bound to?
- Lobe C is bound to H3 and H4 tails
- Lobes A and B are bound to upstream transactivators
what are the enzymatic activities of TAF1?
- kinase
- HAT
- ubiquitin activating/conjugation activity
kinase activity of TAF1
- kinase phosphorylates TFIIF (RAP74)
- significance unclear
HAT activity of TAF1
- adds acetyl groups to amino tail of histones H3 and H4
- loosens chromatin structure
ubiquitin activity in TAF1
- monoubiquinates histone H1
- significance is unclear - loosens chromatin?
what are the functional domains of TAF1 and what are their functions?
- TAND1 and TAND2
- N-terminal domains that contact TBP
- apart of TFIID
drosTAF1
N terminal domain of dTAF1 inhibits transcription by binding to the concave surafe of TBP to mimic the TATAA motif
- acidic activator
acidic activator
- thought to also compete for binding at the same site on TBP
- has been proposed that acidic activators activate transcription by displacing TAF1 binding then are replaced in turn by DNA binding to TBP
why does TFIID not bind to TATAA containing promoters?
TAF1 blocks TBP from binding to TATAA
hTAF9/6 of TFIID mimic which nucleosome structure? what about hTAF12/4b?
- hTAF9/hTAF6 mimic H3/H4
- hTAF12/hTAF4b mimic H2B/H2A
when is TFIID often used?
- used at promoters that lack a TATAA motif
- TBP can function with complexes other than TFIID like in SAGA and PCAF
what are TAFs required for?
- required for developmental processes such as gametogenesis, sperm formation, formation of the neural tube, and specific cell-type differentiation
what percentage of genes have promoters dependent on TFIID?
- 90%
- a group that is largly comprised of housekeeping genes that show basal expression and limited capacity for induction
- the remaining 1-% have promoters that largely rely on SAGA and are often stress inducible
what is the structure of SAGA?
- histone-like octamer
Tra1
- SAGA
- binds upstream activators such as GCN4
GCN5
- SAGA
- contains HAT
DUB
- SAGA
- histone deubiquitinase
Spt3
- SAGA
- binds C-stirrup of TBP
Spt8
- SAGA
- binds N-stirrup of TBP
TBP binding to SAGA
- SAGA is correctly oriented between the 1+ nucleosome and upstream activators
- TBP is positioned over the TATAA motif in the DNA
- TFIIA displaces Spt8 and binds the N-terminal stirrup of TBP
- TBP tightly binds the TATAA which displaces Spt3 binding to the C-stirrup. SAGA leaves
- TBP recruits TFIIB
steps of promoter clearance
- TFIIE and TFIIH are required to melt DNA to allow initiation
- phosphorylation of the CTD of pol 2 by TFIIH at ser5 is required for elongation to begin
- further phosphorylation of the CTD at ser 2 by P-TEFb is required to end abortive initiation
how is CTD involved in RNA processing?
- 7mG capping of 5’ end
- SCAFs recruit splicing factors
- polyadenylation and cleavage of the 3’ end
- elongator complex contains HAT (recruited to inducible promoters)
what percentage of pol 2 promoters are TATAA-less and enriched in GC sequences?
- 76%
- these are expressed in a constitutive manner
what percentage of human pol 2 promoters contain a consensus InR?
46%
what percentage of human pol 2 promoters lack TATAA and the InR?
46%
CpG islands
- regulatory targets, crude level of on/off control
- surround all promoters of constitutively-expressed genes where they are unmethylated (half appear this way)
- also found at the promoters of some tissue-regulated genes (40% of these genes have CpG islands)
how many CpG islands are there in the human genome?
29,000 (45,000 if you include Alu repeats)
- 60% GC compared to 40% for other DNA
what does methylation of a CpG island do?
prevents activation of a promoter within it
- repression is caused by proteins that bind to the methylated CpG doublets
what proteins bind to methylated CpG sequences in vitro?
MeCP1 and MeCP2
- binding of these proteins represses transcription
- MeCP2 binds to the Sin3 repressor complex which contains HDAC
- this removes the acetyl groups from the histones tails and causes chromatin to condense resulting in repression
what is the density of CpG?
- 1/100 bp
- these islands may be 1-2 kb in length and seem to regulate constitutively expressed genes
what organisms have little to no methylation?
- drosophila and dipterans (have a methylase gene)
- nematodes (no methylation)
methylation of CpG islands seems to regulate which type of genes?
constitutive genes
which subunits contain HAT activity?
- TFIID = TAF1
- SAGA = GCN%
- PCAF complex = PCAF subunit
