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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