General Transcription Initiation Factors. Assay for accurate initiation by bacterial RNA polymerase...
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Transcript of General Transcription Initiation Factors. Assay for accurate initiation by bacterial RNA polymerase...
General Transcription Initiation Factors
Assay for accurate initiation by bacterial RNA polymerase
βα
α
σ
Promoter
E. coli RNA polholoenzyme
+
βα
α
σ
initiation
elongationNascent RNA
Run-off transcript,Discrete size
+ +
ββ'
α
α
ββ'
α
α
RNA Pol core
Random transcription by bacterial RNA polymerase core
Promoter
E. coli RNA pol core
+
Random initiation
elongation
Transcripts of random size
ββ'
α
α
ββ'
α
α
ββ'
α
α
ββ'
α
α
ββ'
α
α
ββ'
α
α
Random transcription by eukaryotic RNA polymerase
Promoter
Eukaryotic RNA pol II
+
Random initiation
elongation
Transcripts of random size
Accurate initiation by Euk RNA polymerase II plusfactors in the nucleus
Promoter +
initiation
elongation Nascent RNA
Run-off transcript,Discrete size
+ +
Eukaryotic RNA pol II + Nuclear extract, S-100
GeneralTranscription InitiationFactors
General transcription factors = GTFs• Proteins other than RNA polymerase involved in
transcription – Initiation, Elongation, Termination – Not subunits of purified RNA polymerase.
• GTFs for RNA polymerase II are called TFIIx, where x = A, B, D, …– Can have multiple subunits
• General transcription initiation factors (GTIFs)– Proteins required for specific transcription from
a minimal promoter (core) – Required for RNA polymerase to bind avidly
and specifically to promoters.
Fractionation of nuclear extracts to find GTFsPhosphocellulose column: ion exchange
Template: Adenovirus late promoter
PlusPurifiedPol II
Run-offTranscript:AccurateInitiation at promoter
Polyacrylamide gel separating products of in vitro transcription
Fractionation of nuclear extracts to find GTFsFractionation scheme, DEAE cellulose
Matsui, Segall, Weil, Roeder (1980) JBC 255:11992
PlusPurifiedPol II
Run-offTranscriptAccurateInitiation at promoter
GTIFs for RNA polymerase II
TFIIDTBP }
TAFs
IIB
IIA IIE
IIF
IIH
helicase
protein kinase
TBP
InrIIB IIA Pol IIa
IIFIIE
IIH
CTD of large subunit of Pol II
Recognize core promoter
Targets Pol II to promoter
Modulates helicase
Helicase
CTD protein kinase
Many GTIFs are possible targets for activators of transcription.
TATA Binding Protein = TBP
• TBP binds in the narrow groove of DNA at the TATA box found about 20-25 bp 5’ to the start site for transcription of many (but not all) genes transcribed by RNA polymerase II.
• TBP bends the DNA about 90 degrees.
• TBP alone or with TBP-associated proteins (TAFs) plays an important role in recognizing the core promoter and recruiting RNA polymerase II to the promoter.
TBP bound to DNA
Image from crystal structures, provided by Dr. T. Nixon.
RNA Pol II bound to DNA and general transcription initiation factors
RNA polymerase II from yeast (does not include subunits 4 and 7)
136 Ao
140AoA
o100 thick
Channel 25 Ao
diameter
Groove 25 Ao
wide
flexible end of "thumb"
Channel plus groove could accomodate 20-25 bp of duplex DNA
TFIIA
DNA
TBP
TFIIE
Darst, S.A., A.M. Edwards, E.W. Kubalek & R.D. Kornberg (1991) Cell 66: 121-128.Kornberg, R.D. (1996) Trends in Bioch. Sci. 21: 325-326.
16 resolutionAo
TFIIB
Movie of TBP, then binding to TATA DNA (and bending it), and then TFIIA
binding to TBP-TATA
Thanks to Drs. Song Tan and Tracy Nixon for movies
Sequential Binding
Model for assembly
of preinitiation
complex
-30 +1
TATA Inr
Polymerization of 1st few NTPs and phosphorylation of CTD leads to promoter clearance. TFIIB, TFIIE and TFIIH dissociate, PolII+IIF elongates, and TFIID + TFIIA stays at TATA.
IIB
Eukaryotic RNA polymerase II
TFIID}TBP
TAFs
IIB
IIECTD of large subunit of Pol II
Pol IIa
or TBP
IIA
IIF
helicase
protein kinase
IIH
TATA Inr
IIA Pol IIa
IIFIIE
preinitiation complex
TATA Inr
IIAIIB Pol IIa
IIFIIE
ATP hydrolysis
initiation complex, DNA melted at Inr
IIH
IIH
= PIC
Activated PIC
Direct Binding of
Holoenzyme model for
assembly of the
preinitiation complex
TFIID}TBP
TAFs
-30 +1
TATA Inr
TBP
preinitiation complex
ATP hydrolysis
initiation complex, DNA melted at Inr
or
TATA Inr
IIBPol IIa
IIF
IIH
IIESRB
IIB
TATA Inr
Holoenzyme
Pol IIa
IIF
IIH
IIE
IIA
SRB
activator?
IIBPol IIa
IIF
IIH
IIESRB
IIATATA
IIA ?
= PIC
Activated PIC
Other proteins involved in transcription and regulation
• In addition to RNA polymerase II and GTIFs:• Proteins required for regulation, e.g.
– Gal11: regulation of the GAL operon– Rgr1: resistance to glucose repression
• Srb proteins– Yeast strains with truncations in the CTD of the large
subunit of RNA polymerase B are cold-sensitive– SRB genes: when mutated, suppress the phenotype of
CTD deletions– Extragenic suppressors: implicated in RNA polymerase
function
RNA polymerase II Holoenzyme and Mediator
• Holoenzyme– RNA polymerase II + (TFIIB, E, F, H )+ (Srb2, 4, 5, 6) +
(Rgr1, Gal11, others)– Correct initiation in presence of TBP (TFIID)– Responds to transcriptional activators
• Mediator– Complex needed for a response to transcriptional
activators by purified RNA Pol II plus GTFs– Yeast Mediator has 20 subunits, including Srb2, 4, 5, 6;
Srb7, Rgr1, Gal11, Med 1, 2, 6, 7, Pgd1, Nut 1, 2, and others
• RNA Pol II + Mediator (+ some GTIFs?) = Holoenzyme
Expanding the functions of RNA polymerase
Polymerase Trans-cribe
Start atPromoter
Respond toActivator
RNA Pol II Yes No NoRNA Pol II +GTIFs
Yes Yes No
RNA Pol IIholoenzyme+ GTIFs
Yes Yes Yes
Parallels between initiation
pathway in prokaryotes and
eukaryotes
From Eick et al. (1994)Trends in Genetics 10: 292-296
GTFs for RNA polymerase I
Eukaryotic RNA polymerase I
UBF1
SL1}
TBP
Pol I
GTFs for RNA polymerase III
IIIAIIICIIIC
}TFIIIB
TBP
Pol IIIEukaryotic RNA polymerase III