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Molecular Genetics > Eukaryotic Transcription 2 & 3 > Flashcards

Eukaryotic Transcription 2 & 3 Flashcards

1
Q

TBP

A
  • a component of the positioning factor that is required for each type of RNA polymerase to bind its promoter
  • mass of 800 kD
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2
Q

what is the TBP for RNA polymerase 2?

A
  • TF2D
  • consists of TBP and 11-14 TAFs (TBP associated factors
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3
Q

how does TBP bind to DNA?

A
  • 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
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4
Q

what determines the polarity of transcription?

A
  • TBP
  • based on the asymmetry in the TATAA sequence itself and interactions with TF2B
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5
Q

how does TBP insert a kin in DNA?

A

inserts 2 phenylalanines

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6
Q

where does TF2B bind?

A
  • binds to C- terminal stirrup
  • plays a critical role in determining the start site for transcription in association with TBP
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7
Q

B-linker helix

A
  • apart of TF2B
  • melts the promoter 20 bp upstream from TATAA
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8
Q

B-reader helix

A
  • apart of TFIIB
  • contacts -8 of the initiator element
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9
Q

what does the B-reader helix and B-reader loop do?

A
  • contact the 5 end of the nascent transcript
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10
Q

B-reader loop

A

contacts position -1

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11
Q

the binding of what protein is the first step in initiation?

A
  • 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
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12
Q

how is TBP delivered to the promoter?

A

by TFIID

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13
Q

TAFs 1 and 7

A

bind InR and other downstream elements

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14
Q

TAFs 11 and 13

A

block c-stirrup of TBP

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15
Q

TAF 1

A

blocks DNA binding cleft and n-stirrup of TBP

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16
Q

what is the process of loading TBP onto DNA?

A
  • 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
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17
Q

where is each lobe bound to?

A
  • Lobe C is bound to H3 and H4 tails
  • Lobes A and B are bound to upstream transactivators
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18
Q

what are the enzymatic activities of TAF1?

A
  • kinase
  • HAT
  • ubiquitin activating/conjugation activity
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19
Q

kinase activity of TAF1

A
  • kinase phosphorylates TFIIF (RAP74)
  • significance unclear
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20
Q

HAT activity of TAF1

A
  • adds acetyl groups to amino tail of histones H3 and H4
  • loosens chromatin structure
21
Q

ubiquitin activity in TAF1

A
  • monoubiquinates histone H1
  • significance is unclear - loosens chromatin?
22
Q

what are the functional domains of TAF1 and what are their functions?

A
  • TAND1 and TAND2
  • N-terminal domains that contact TBP
  • apart of TFIID
23
Q

drosTAF1

A

N terminal domain of dTAF1 inhibits transcription by binding to the concave surafe of TBP to mimic the TATAA motif
- acidic activator

24
Q

acidic activator

A
  • 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
25
Q

why does TFIID not bind to TATAA containing promoters?

A

TAF1 blocks TBP from binding to TATAA

26
Q

hTAF9/6 of TFIID mimic which nucleosome structure? what about hTAF12/4b?

A
  • hTAF9/hTAF6 mimic H3/H4
  • hTAF12/hTAF4b mimic H2B/H2A
27
Q

when is TFIID often used?

A
  • used at promoters that lack a TATAA motif
  • TBP can function with complexes other than TFIID like in SAGA and PCAF
28
Q

what are TAFs required for?

A
  • required for developmental processes such as gametogenesis, sperm formation, formation of the neural tube, and specific cell-type differentiation
29
Q

what percentage of genes have promoters dependent on TFIID?

A
  • 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
30
Q

what is the structure of SAGA?

A
  • histone-like octamer
31
Q

Tra1

A
  • SAGA
  • binds upstream activators such as GCN4
32
Q

GCN5

A
  • SAGA
  • contains HAT
33
Q

DUB

A
  • SAGA
  • histone deubiquitinase
34
Q

Spt3

A
  • SAGA
  • binds C-stirrup of TBP
35
Q

Spt8

A
  • SAGA
  • binds N-stirrup of TBP
36
Q

TBP binding to SAGA

A
  • 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
37
Q

steps of promoter clearance

A
  • 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
38
Q

how is CTD involved in RNA processing?

A
  • 7mG capping of 5’ end
  • SCAFs recruit splicing factors
  • polyadenylation and cleavage of the 3’ end
  • elongator complex contains HAT (recruited to inducible promoters)
39
Q

what percentage of pol 2 promoters are TATAA-less and enriched in GC sequences?

A
  • 76%
  • these are expressed in a constitutive manner
40
Q

what percentage of human pol 2 promoters contain a consensus InR?

A

46%

41
Q

what percentage of human pol 2 promoters lack TATAA and the InR?

A

46%

42
Q

CpG islands

A
  • 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)
43
Q

how many CpG islands are there in the human genome?

A

29,000 (45,000 if you include Alu repeats)
- 60% GC compared to 40% for other DNA

44
Q

what does methylation of a CpG island do?

A

prevents activation of a promoter within it
- repression is caused by proteins that bind to the methylated CpG doublets

45
Q

what proteins bind to methylated CpG sequences in vitro?

A

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

46
Q

what is the density of CpG?

A
  • 1/100 bp
  • these islands may be 1-2 kb in length and seem to regulate constitutively expressed genes
47
Q

what organisms have little to no methylation?

A
  • drosophila and dipterans (have a methylase gene)
  • nematodes (no methylation)
48
Q

methylation of CpG islands seems to regulate which type of genes?

A

constitutive genes

49
Q

which subunits contain HAT activity?

A
  • TFIID = TAF1
  • SAGA = GCN%
  • PCAF complex = PCAF subunit

Molecular Genetics (28 decks)

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