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