Ch 11. General Transcription Factors in Eukaryotes.

Ch 11. General Transcription Factors in Eukaryotes

General transcriptional factors in eukaryotes

RNA pol + promoter DNA + ? transcription

DNA-Protein interaction

염기쌍 간의 수소결합에 영향을 주지 않는 상태에서 염기의 가장자리와 비공유결합을 통해 결합

Transcription in Eukaryotes

• 다양한 RNA 중합효소 (Pol I,II,III)

• 보편전사인자 (general transcription factors)

• 조절성 DNA 염기서열 : 거리 , 위치 , 다양성 (enhancer)

• DNA 가 응축된 상태에서 이루어진다 (Chromatin remodeling)

General Transcription Factors

• 프로모터와 RNA 중합효소의 결합을 유도한다

• DNA 의 두 가닥을 벌려 전사개시를 돕는다

• 전사개시 후 프로모터로부터 RNA 중합효소를 분리시킨다

RNA 중합효소 및 기타 조절인자들과 더불어 전사개시 복합체

(transcription initiation complex) 를 형성한다

Formation of Class II pre-initiation complex RNA polymerase II,

TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH

in vitro assembly of pre-initiation complex using purified complex

Footprinting the DA and DAB complex

Footprinting theDABPolF complexes

Model for formation of the DABPolF complex

Structure and Function of TFIID

• TFIID; TBP + 8-10 TBP associated factors (TAFIIs)

• TBP; 37 kDa, TATA-box binding protein highly conserved in all eukaryotes (>80% identity) • SL1: selectivity factor for Pol I, TBP + 3 TAF's TFIIIB: transcription factor for Pol III, TBP + 2TAF's

• TFIID bind to minor groove methylations in TATA box interfere TFIID binding

Substitution of C for T, I for Ano effect on TBP binding to MLP(major late promoter of adenovirus) TATA minor groove binding

Structure of TBP-TATA complex;

Binding of TBP to DNA forces DNA bending open promoter complex

TBP functions in

pol II promoters with TATA pol II with TATA-less promoters

pol I & pol III promoters; Ts mutants

Versatility of TBP

Drosophila TFIID assembled from the products of cloned genes(Genes for 8 proteins co-immunoprecipitated with TBP were cloned)

TBP-associated factors (TAFIIs)

TAFIIs :

• Interact with promoter• Interact with activator

Difference between TBP and TFIID action

TAFIIs facilitate transcription from promoters with Inr & DPE

Role of TAFII: 1. Interactions with promoters

TAFIIs interacting with Hsp70 promoter

UV-cross-linking exp.with DNA (BrdU) labeled with 32P

DNaseI Footprinting of hsp70 promoter

TAFII250 and TAFII150 cooperate

in binding to the initiator & DPE

TATA-less promoters ? TAFIIs helps anchoring TBP (TFIID)

DPE

DPE

DPE

TBP alone cannot respond to Sp1 but TFIID can do

TAFs = co-activators

Sp1; GC box-binding activator

Role of TAFIIs: 2. Transcription stimulation by activators

Model for transcriptionactivation by activators

different transcription factors work with different TAFs

TAF250: assembly factor ?

enzyme activity of TAFs TAF250: HAT & kinase activity

Exceptions in the generality of TFIID

• TAFIIs are not universally required for PolII promoters.

• Even TBP is not universal ; TRF1, TLF (TBP-like factor)

• Some promoter-specific TBP alternatives exist

• TFTC (TBP-free TAFII-containing complex)

• NC2 (Negative cofactor 2) ; – Stimulate DPE-containing promoter

– Repress TATA box containing promoter by blocking the binding of TFIIB and TFIIA

TAFII17 is a part of transcription adaptor complex, SAGA• (SPTs, ADAs, & GCN5, & histone acetyltransferase)• mediate the effects of transcription activator proteins

TFIIDs are heterogeneous in their TAF composition

TRF1 (TBP-related factor 1)• in developing neural tissue in Drosophila • bind to TFIIA and TFIIB• TRF-associated factors, nTAFs (neural TAFs)• two promoters in tudor gene• TC box: -22 -33, ATTGCTTTTCTT

TLF (TBP-like factor) ; TATA-less promoter

TFTC (TBP free TAFII-containing complex)

TFIID

TFTC

Functions of other TFs

TFIIA: • dimmer of 32 & 13.5 kDa subunits• not required for pre-initiation complex in vitro, but, essential in vivo• TFIIA as a TAFII? binds to TBP & stabilize binding between TFIID & promoter

TFIIB: • 35 kDa monomer, • essential for RNA pol binding• serves as a linker between TFIID and Pol/F complex ; 2 domains• target of common activator – facilitate binding of TFIIB to the pre-initiation complex• Rate-limiting step for transcription

TFIIF: RAP30 (similar to bacterial factor) & RAP70, interaction with RNA pol & DAB reduce non-specific binding to DNA

TFIIE: tetramer of 56 kDa & 34 kDa, p56 contains a Zn-finger binds after Pol/F joins the complex

TFIIH: 4 with protein kinase activity (phosphorylation of CTD) + 5 with DNA helicase/ATPase activity

Pre-initiation complex

Phosphorylation of pre-initiation complex

IIA

IIO

TFIIH

P

ATP

TFIIH phosphorylates RNA polymerase II

TFIIE stimulates phosphorylation

+TFIIE

TFIIH phosphorylates the CTD of Polymerase II

( TFs + RNA pol II + -32P ATP )

Helicase activity assay of TFIIH in vitro ; melting, further unwinding & releases RNA Pol from promoter

Lane 1; heat-denatured templateLane 2; no proteinLane 3; TFIIH + no ATPLanes 4-5; increasing amount of TFIIH +ATP

(RAD25)

permissive

Non-permissive

• pol II transcription requires ATP hydrolysis two candidates: CTD phosphorylation X DNA helicase activity

TFIIE & TFIIH are not required for abortive transcription of trinucleotides not for initiationTFIIH is required for complete unwinding of promoters

• TFIIH for elongation ? initiation of transcription up to 17 mer without TFIIE & TFIIH (using supercoiled template) elongation in the presence or absence of TFIIE & TFIIH no difference in elongation reaction

TFIIE & TFIIH are required for promoter clearance.

Model for the Transcription initiation, promoter clearance, and elongation

Pre-initiation complex

Elongation factors (IIS)

Minimize Polymerase pausing enhance transcription elongation

Proofreading by RNA polymerase II

(arrest)

RNA polymerase II holoenzyme

Holoenzyme;

core enzyme (RNA polymerase)+ Transcription factors except TFIID + SRB proteins (suppressor of RNA polymerase B) + mediators+ Swi/Snf complex, etc

Reconstituted a yeast holoenzyme with 58 subunits (Young, 1998)

SL1 (TIF-IB) : core-binding factor

Species specificity

Upstream-binding factor (UBF) ; assembly factor

UBF bind to the promoter UPE A site /core element

SL1 facilitate the binding ; extend to B site

UPE

SL1 and UBF act synergistically to activate transcription from rRNA promoter;

• SL1 needed for basal activity• UBF enhance transcriptional activity by binding to the UPE, but is not necessary.

(deletion of UPE)

Immunodepletion of TBP

• inhibits SL1 activity• restored by addition of SL1 • but, not by adding back TBP

What other factors? : TAFIs

SL1 ; TBP + three TAFIs

TBP is identical, but TAFs are completely different ; • Species- specific• compete for TBP ; SL1 vs. TFIID

Class III factors

TFIIIA :

• 5S rRNA• First eukaryotic transcription factor • 9 Zn-finger motif interacting with major groove Anti-TFIIIA

TFIIIB &C : tRNA

Box A box B

tRNA

TFIIIB

TFIIIC

Class III Factors on tRNA promoter

?

TFIIIC and A (assembly factor) are required for the TFIIIB binding to the promoter of 5S rRNA

TFIIIB recruit pol III and continue to promote further transcription

TFIIIC cause conformational change in TFIIIB - tight bound to DNA

Transcription Factors forming pre-initiation complex on TATA-less promoters of three classes

Role of TBP in TATA-less promoter

• Bring the remaining factor to the complex• Organizing preinitiation complex• Specificity of TBP is governed by TAFs