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 the DABPolF complexes

Model for formation of the DABPolF complex

Structure and Function of TFIID TFIID; TBP TBP associated factors (TAF II s) 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 A no 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 (TAF II s)

TAF II s : Interact with promoter Interact with activator

Difference between TBP and TFIID action TAF II s facilitate transcription from promoters with Inr & DPE Role of TAF II : 1. Interactions with promoters

TAF II s interacting with Hsp70 promoter UV-cross-linking exp. with DNA (BrdU) labeled with 32 P

DNaseI Footprinting of hsp70 promoter TAF II 250 and TAF II 150 cooperate in binding to the initiator & DPE

TATA-less promoters ? TAF II s helps anchoring TBP (TFIID) DPE

TBP alone cannot respond to Sp1 but TFIID can do TAFs = co-activators Sp1; GC box-binding activator Role of TAF II s: 2. Transcription stimulation by activators

Model for transcription activation 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 TAF II s 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 TAF II -containing complex) NC2 (Negative cofactor 2) ; –Stimulate DPE-containing promoter –Repress TATA box containing promoter by blocking the binding of TFIIB and TFIIA

TAF II 17 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 TAF II -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 TAF II ? 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 +  - 32 P ATP )

Helicase activity assay of TFIIH in vitro ; melting, further unwinding & releases RNA Pol from promoter Lane 1; heat-denatured template Lane 2; no protein Lane 3; TFIIH + no ATP Lanes 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 initiation TFIIH 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? : TAF I s

SL1 ; TBP + three TAF I s 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