TRANSCRIPTIONTRANSCRIPTIONand and

TRANSCRIPTION FACTORSTRANSCRIPTION FACTORS22.10. 201322.10. 2013

CEREN GONEN KORKMAZ, PhD

Transcription in Transcription in EukaryotesEukaryotes

RNA polymerase I transcribes rRNA RNA polymerase I transcribes rRNA RNA polymerase II transcribes mRNARNA polymerase II transcribes mRNA RNA polymerase III transcribes RNA polymerase III transcribes tRNA and other small RNAs. tRNA and other small RNAs.

Animal RNA Polymerases Animal RNA Polymerases Animal DNA-dependent RNA Polymerases Animal DNA-dependent RNA Polymerases Class α-amantin sensitivity Major Products Class α-amantin sensitivity Major Products I Insensitive I Insensitive

rRNA rRNA II Low Conc. (1-10 nM) hnRNA II Low Conc. (1-10 nM) hnRNA III High conc. III High conc.

tRNA, 5S RNA tRNA, 5S RNA

and small RNAs and small RNAs

All have in common 2 large subunits and a number of smaller All have in common 2 large subunits and a number of smaller subunits, as well as being zinc metalloenzymes. subunits, as well as being zinc metalloenzymes.

TATA box is a septamer (TATAAAA ) at -25 and is involved in positioning the enzyme for correct initiation.

CAAT box (CCAATCT) is at –75 and is recognized by a large group of transcription factors and plays a strong role in determining the efficiency of the promoter.

GC box is at -90 contains the sequence GGGCGG and is recognized by the factor SP1.

Initiator has a general form Py2ANT/APy2 and has the simplest possible form recognizable by RNA polymerase II.

Elements combination in type II Promoters

Enhancer; Dehancer; Silencer; Upstream Activating Sequences (UAS)

More than 10 Subunits to form RNA polymerase II

Subunits of RNA Subunits of RNA polymerase IIpolymerase II

Core-enzyme with the Core-enzyme with the active siteactive site RPB1 (RPB1 (´-like) binds ´-like) binds DNADNA

RPB2 (RPB2 (-like) binds NTP-like) binds NTP RPB3 (RPB3 (-like) assembly -like) assembly factorfactor

Common subunitsCommon subunits RPB5, 6 and 8 common RPB5, 6 and 8 common to RNAPI-IIIto RNAPI-III

Common functions?Common functions? Three non-essential: Three non-essential: RPB4, 7 and 9RPB4, 7 and 9

´

DNA-bindingNTP-binding

Prokaryot

Eukaryot

Structure 1999 - 2000Structure 1999 - 2000

ß

ß´

•1990: 2D crystal - low resolution•1999: First 3D-structure published

Yeast yRNAPII 6Å resolutionyRNAPII + DNA/RNA low resolutionTaq RNAPII high resolution 3.3Å

• 2000: 3D crystal - high resolution 10 subunit yRNAPII - 3Å resolution

2.8Å structure(Science)

Structure 1999Structure 1999

Secondary channel

ASC

yRNAPII (closed)Taq RNAP (open)

Armlukket

Finger

DNAdownstream

New RNA exitsHenglet domene

Hylle(shelf)

ASC = active site channel

Arm (ß2)open

DNAupstream

JawsJaws

NTP

ASC: 25Å channel through ASC: 25Å channel through the enzymethe enzyme

yRNAPII

Yeast Yeast RNAPIIRNAPII

Notable features: A pair of jaws that appear to grip DNA downstream of the active center. A clamp on the DNA nearer the active center may be locked in the closed position by RNA, accounting for the great stability of transcribing complexes. A pore in the protein complex beneath the active center may allow entry of substrates for polymerization and exit of the transcript during proofreading and passage through pause sites in the DNA.

Opening and closing of Opening and closing of RNAPIIRNAPII

Open RNAP during formation of Open RNAP during formation of PICPIC

RNAP closes during promoter RNAP closes during promoter clearance and transition to clearance and transition to

TECTEC RNAP opens and becomes RNAP opens and becomes

destabilised during destabilised during terminationtermination

Conformational changes Conformational changes during during

the transcription cyclethe transcription cycle

Rearranged to a very stable TEC (transcription elongation complex) that can move trough 104-106 bp without dissociation.Footprint reduced (35 bp). Euk: phosphoryl. of CTD and association with elong.factors

open

open

open

closed

Large footprint (70-90 bp) caused by DNA wrapped around RNAP”Abortive initiation” may happen in this state (RNA <10 nt)

Destabilised again during termination

Transition

CTD - C-terminal domainCTD - C-terminal domain Conserved tail on the largest Conserved tail on the largest subunit: (YSPTSPS)nsubunit: (YSPTSPS)n Yeast n = 26, humans n = 52Yeast n = 26, humans n = 52

Unique for RNAPIIUnique for RNAPII Essential function in vivoEssential function in vivo

>50% lethal>50% lethal Reversible phosphorylation on Ser Reversible phosphorylation on Ser and Tyrand Tyr

CTDs phosphorylation CTDs phosphorylation changes during the transcription changes during the transcription

cyclecycle Reversible phosphorylation on both Ser and TyrReversible phosphorylation on both Ser and Tyr Creates different forms of RNAPIICreates different forms of RNAPII

RNAPIIORNAPIIO - hyperphosphorylated (Mr=240k) - hyperphosphorylated (Mr=240k) ≈ ≈ 50 50 phosphphosphatatee ( (oneone p peer repeat)r repeat) Abl-Abl-phoshorylatedphoshorylated in vitro ≈30 in vitro ≈30 phosphatephosphate

RNAPIIARNAPIIA - without phosphate (Mr=214k) - without phosphate (Mr=214k) RNAPIIBRNAPIIB - with CTD deleted - with CTD deleted

Function of RNAPIIA ≠ RNAPIIOFunction of RNAPIIA ≠ RNAPIIO PIC assembly: only non-phosphorylated RNAPIIAPIC assembly: only non-phosphorylated RNAPIIA Elongation compleElongation complexx: only hyperphosphor. RNAPIIO: only hyperphosphor. RNAPIIO

Phosphorylation status changes during the transcription Phosphorylation status changes during the transcription cyclescycles

CTD-phosphorylation CTD-phosphorylation changes changes

during the transcription during the transcription cyclecycle

PPPPPPPPPPP

PPPPPPPPPPP

CTDP

RN AP IIAcccl

RN AP IIA

RN AP IIO

PPPPPPPPPPP

CTDK

RN AP IIA

RN AP IIO

PIC assembly

Clean until new assembly

dephosphorylation

free

initiation

phosphorylation

Elongation

CTD-binding proteinsCTD-binding proteins SRBs - supressors of RNA pol. BSRBs - supressors of RNA pol. B GTFsGTFs

TBPTBP TFIIF (74 kDa)TFIIF (74 kDa) TFIIE (34 kDa)TFIIE (34 kDa)

CTDs functionCTDs function InitiInitiationation

-Role in Recruitment of RNAPII to promoter-Role in Recruitment of RNAPII to promoterinterainteraction withction with GTF (TBP, TFIIF GTF (TBP, TFIIF andand TTFIIE)FIIE)

Promoter clearancePromoter clearance Def: The process whereby RNAPII undergoes Def: The process whereby RNAPII undergoes the transition to hyperphosphorylated the transition to hyperphosphorylated elongation moduselongation modus

HypothesisHypothesis: CTD phosphorylation disrupts : CTD phosphorylation disrupts interactions and RNAPII gets free from PICinteractions and RNAPII gets free from PIC

HypothesisHypothesis: CTD phosphorylation creates : CTD phosphorylation creates novel interactions with elongation factorsnovel interactions with elongation factors

• TBP: TATA-binding protein• TAFs: TBP-associated factors• TFIID protects a region extending farther upstream• TFIIA activates TBP by relieving the repression that is caused by the TAFs • TFIIB binds adjacent to TBP and TATA box• TFIIF consists of two subunits. The larger subunit has an ATP-dependent DNA helicase activity and the small one contacts the core polymerase.• TFIIE and TFIIH are required for promoter clearance to allow RNA polymerase to commence movement away from the promoter.

How does RNA polymerase II initiate transcription?

TFIID

• TFIIH has several activities, including an ATPase, a helicase, and a kinase activity that can phosphorylate the CTD tail of RNA polymerase II; it is also involved in repair of damage to DNA.

Most of the TFII factors are released before RNA polymerase II leaves the promoter.

Phosphorylation of the CTD by the kinase activity of TFIIH may be needed to release RNA polymerase to start transcription.

TBP is a universal factor

GTFs and PIC assemblyGTFs and PIC assembly General transcription factors (GTFs)General transcription factors (GTFs)

make RNAPII capable of selective initiation make RNAPII capable of selective initiation in vitroin vitro Studied in several systemsStudied in several systems

Human (HeLa)Human (HeLa) RRatat DrosophilaDrosophila yeast (Saccharomyces cerevisiae)yeast (Saccharomyces cerevisiae)

Highly conservedHighly conserved RNAPII+GTS = ca. 30 polypeptidesRNAPII+GTS = ca. 30 polypeptides

≈ ≈ 2 MDa2 MDa

TBP

TFIIB

TFIIA

TFIIE

TFIIF

TFIIH

+ =Correctinitiationof trscrin vitro

PIC

Assembly of PIC -Assembly of PIC -the preinitiation the preinitiation

complexcomplex NucleationNucleation: : TFIID+TATA form an “initial committed complex”TFIID+TATA form an “initial committed complex” IBP/TAFs + INR may also initiate PIC-assemblyIBP/TAFs + INR may also initiate PIC-assembly For snRNAs: PSE+SNAPcFor snRNAs: PSE+SNAPc Common: a core sequence is recognized by a Common: a core sequence is recognized by a seq.spes.GTFseq.spes.GTF

LinkLink:: initial complex recognized by TFIIBinitial complex recognized by TFIIB With TFIIB bound, the complex becomes accessible With TFIIB bound, the complex becomes accessible to RNAPIIto RNAPII

RNAPII recruitment:RNAPII recruitment: Assembly of RNAPII assisted by TFIIFAssembly of RNAPII assisted by TFIIF Minimal initiation complex formedMinimal initiation complex formed

Maturation Maturation to complete transcription competent to complete transcription competent PICPIC::

Minimal initiation complex (DABF-pol) NOT Minimal initiation complex (DABF-pol) NOT tr.competenttr.competent

Recruitment of TFIIH and TFIIE necessaryRecruitment of TFIIH and TFIIE necessary This step is unique for RNAPIIThis step is unique for RNAPII

PIC assembly - linear pathway

TAFsIID

TBPTFIIBTFIIA RNAPII

TFIIFTFIIHTFIIE

TATA INR

TFIIB

TBP or TFIID + TFIIA

TFIIF-RNAPIIA

TFIIE

TFIIH

DNA

CTD

Linear PIC-Linear PIC-assemblyassembly

PIC assembly - linear pathway

TAFsIID

TBPTFIIBTFIIA RNAPII

TFIIFTFIIHTFIIE

TATA INR

TFIIB

TBP or TFIID + TFIIA

TFIIF-RNAPIIA

TFIIE

TFIIH

DNA

CTD

AlternativeNucleation events Nucleation

Link

RNAPII recruitment

MaturationHoloenzyme

2-stepalternative

TBPTBP [TFIID] function [TFIID] function Binds TATA - main sequence recognition Binds TATA - main sequence recognition

event during PIC assemblyevent during PIC assembly BindBinds manys many TATA-like TATA-like factorsfactors minor groove minor groove ccontaontacctt BindBinds ass as monomer monomer

Affinity of TBP for TATA contributes to Affinity of TBP for TATA contributes to promoter strengthpromoter strength

Binds also several other polypeptidesBinds also several other polypeptides aacctivatortivatorss (Sp1, Tax1, E1A) (Sp1, Tax1, E1A) TAFs (dTAF110, dTAF40)TAFs (dTAF110, dTAF40) GTFs (TFIIB, TFIIA)GTFs (TFIIB, TFIIA) inhibitorinhibitorss

TBP = universal TF involved in all three TBP = universal TF involved in all three pol syst.pol syst. TBP iTBP inn SL1, TFIID, TFIIIB SL1, TFIID, TFIIIB

DNA

Other factors

N

TBP-structureTBP-structure Subunit and cDNA-structureSubunit and cDNA-structure

TFIID = TBP + multiple TAFsTFIID = TBP + multiple TAFs mammalian TFIID: 750 kDa (II), 300 kDa (III) and 200 kDa (I)mammalian TFIID: 750 kDa (II), 300 kDa (III) and 200 kDa (I) TBP only a small core in the TFIID complex (in the exam)TBP only a small core in the TFIID complex (in the exam)

human 38 kDa, yeast 27 kDa, Arabidopsis 22 kDahuman 38 kDa, yeast 27 kDa, Arabidopsis 22 kDa TBP=N-term.divergent domain + C-term.TBP=N-term.divergent domain + C-term.conservedconserved domain domain

C-term.domC-term.domain ain 180aa 180aa lenghtlenght Shows all essential functionsShows all essential functions

N-term.N-term.domaindomain FunFunction is unclearction is unclear

3D: saddle-structure3D: saddle-structure Twofold symmetry and the form of a saddleTwofold symmetry and the form of a saddle Concave inside binds DNA in minor groove through a 10-Concave inside binds DNA in minor groove through a 10-

stranded antiparallel stranded antiparallel -sheet -sheet Convex surface binds other GTFs via 4 Convex surface binds other GTFs via 4 -helixes-helixes Loop on each side with Phe side-chains interLoop on each side with Phe side-chains interchchalating in DNAalating in DNA

TAFs

TBP

TBPs saddle-structureTBPs saddle-structure

Concaveinside

Convexoverside

3D: saddle-structureTwofold symmetry forms of a saddle.Concave inside binds DNA in minor groove through a 10-stranded antiparallel -sheet Convex surface binds other GTFs via 4 -helixesLoops on each side with Phe side-chains interchalating in DNA

Loops

TBPs effect on DNATBPs effect on DNA DNA-structure is distorted upon TBP DNA-structure is distorted upon TBP bindingbinding

Effect?Effect? Upstream and downstream elements come closerUpstream and downstream elements come closer IInncompatible compatible with nu with nuccleosome structureleosome structure

Not like this .. but this way

TBP involved in multiple TBP involved in multiple complexcomplexeses

RNAPI RNAPII RNAPIII

10x more TBP in a cell than TAFs

TLF - TBP like factorTLF - TBP like factor

TBP TLFTRF1

2 TBP like proteinsin multicellular organisms

TLPTRF2TRFTRP

Drosophila

≈

TBP top view

TBP bottom view

TBP specificTLF specific

Pol.III factorOnly?

TLF fails to bind TATATLF fails to bind TATA TLF retained interaction with TLF retained interaction with

TFIIA, TFIIB and RNAPIITFIIA, TFIIB and RNAPII

TFIIBTFIIB Functions in PIC-assembly as adaptor - a Functions in PIC-assembly as adaptor - a molecular bridge that couples IID-TATA molecular bridge that couples IID-TATA with RNAPIIwith RNAPII TFIIB recognizes the distorted TBP-TATA complexTFIIB recognizes the distorted TBP-TATA complex TFIIB mediates RNAPII bindingTFIIB mediates RNAPII binding

İnteraİnteracts also withcts also withTFIIFTFIIF Function in initiation: Function in initiation: ““Measures” distance TATA - TSSMeasures” distance TATA - TSS

+1TFIIB

TFIIB cont.TFIIB cont. TFIIB also contact point for activatorsTFIIB also contact point for activators

VP16, Steroid hormone receptVP16, Steroid hormone receptorsors, fushi tarazu, TAF40), fushi tarazu, TAF40)

+1TFIIB

TFIIB-structureTFIIB-structure C-terminal core C-terminal core domain (TFIIBc)domain (TFIIBc)

DNADNA contact before and contact before and after after TBPTBP

N-terminaN-terminal l essential essential for RNAPII contactfor RNAPII contact

MediMediates contact withates contact with RNAPII-TFIIF complexRNAPII-TFIIF complex

+1TFIIBC-term core

N

TFIIBc structureTFIIBc structure

TFIIB

TBP

TSSSide view rel to TBP

Two globular repeatscontact DNA before and after TBP

TFIIATFIIA ControversialControversial

not essensial in vitro with TBP and purified not essensial in vitro with TBP and purified componentscomponents

required with TFIID and less purified systemrequired with TFIID and less purified system FunctionFunction

counteracts repressors associated with TFIID counteracts repressors associated with TFIID (Dr1, topoI, MOT1)(Dr1, topoI, MOT1)

Stabilizes the TBP-TFIIB complexStabilizes the TBP-TFIIB complex TBP-mutant TBP-mutant with with defe defecct TFIIA interat TFIIA interactionction Able to enter the PIC assembly on all steps Able to enter the PIC assembly on all steps after TFIID bindingafter TFIID binding

Required for activator-responseRequired for activator-response

Structure TFIIAStructure TFIIA human/drosophila heterotrimer: 37 + 19 + human/drosophila heterotrimer: 37 + 19 + 13 kDa (13 kDa ()) BBothoth and and produ producctt of of same gensame genee – proteoly – proteolysis product sis product ??

yeast: heterodimer: 32 + 13 kDayeast: heterodimer: 32 + 13 kDa TOA1 32kDa (homolog human TOA1 32kDa (homolog human og og ) essensiell) essensiell TOA2 13 kDa essensiellTOA2 13 kDa essensiell

Antirepression requires Antirepression requires Activation requires Activation requires 3D 3D two domains form an L-formed two domains form an L-formed structurestructure TOA1 TOA1 and and TOA TOA22 L N

C

Yeast TOA1

Human

Human ß

TFIIA structureTFIIA structure

TFIIA

TBPC-terminal ß-barrelcontacts DNA and TBP

N-terminal4-helix bundle.Probably Activator contact

TFIIA - DNA-interactionTFIIA - DNA-interaction Interaction with DNA upstream TATAInteraction with DNA upstream TATA C-terminal C-terminal -barrel -barrel both TBP- and both TBP- and DNA-interactionDNA-interaction

TFIIAs N-terminal TFIIAs N-terminal -helix structure -helix structure generates an interaction domain generates an interaction domain necessary for activator contactnecessary for activator contact

TFIIA and TFIIB bind on opposite TFIIA and TFIIB bind on opposite sides of DNA without collisionsides of DNA without collision

TBPs convex surface still exposed TBPs convex surface still exposed for other interactions for other interactions

TFIIA-TBP-TFIIB: place TFIIA-TBP-TFIIB: place for allfor all

TFIIA

TBP

TFIIB

TFIIFTFIIF ( (also calledalso called RAP RAP RNAPII-RNAPII-ass. faktorass. faktor))

Structure:Structure: Heterodimer iHeterodimer in high eukaryotesn high eukaryotes

RAP30 + RAP74 (Mw: 26 + 58 kDa)RAP30 + RAP74 (Mw: 26 + 58 kDa) S.cer.TFIIF heterotrimer: 105, S.cer.TFIIF heterotrimer: 105,

54, 30 kDa54, 30 kDa Distinct feature: function both in Distinct feature: function both in

initiation and elongationinitiation and elongation Initiation - helps in the Initiation - helps in the

recruitment of RNAPIIrecruitment of RNAPII Stabil assStabil association ofociation of RNAPII RNAPII

withwith TFIIF TFIIF Initiation: a role in recruitment Initiation: a role in recruitment

of TFIIE+TFIIHof TFIIE+TFIIH Elongation: enhance catalytic Elongation: enhance catalytic

velocity of RNAPIIvelocity of RNAPII

TFIIB

TFIIF

TFIIFTFIIF = heterotetramer (RAP30 = heterotetramer (RAP3022 RAP74RAP7422))

RAP30: Two RAP30: Two -related domains-related domains RAP30 crosslinks DNA RAP30 crosslinks DNA betweenbetween TATA TATA andand TSS TSS

RAP30 bindRAP30 binds alsos also TFIIB TFIIB RAP74: glob.N - central charged region - RAP74: glob.N - central charged region - glob.Cglob.C Stimulates elongationStimulates elongation

RAP74 bindRAP74 binds s RNAPIIRNAPII RAP74+30 RAP74+30 can removecan remove RNAPII RNAPII from unspecific from unspecific DNADNA RAP74 RAP74 crosslinks DNA crosslinks DNA betweenbetween TATA TATA andand TSS TSS

TAFII250 TAFII250 likelike kinase kinase Phosphorylation activates Phosphorylation activates TFIIFTFIIF

30 30

74 74

RNAPIIDNATFIIB

RNAPIIDNA

P P P

P

TFIIETFIIE structurestructure

heterotetramer heterotetramer 2222: 34 + 56 kDa: 34 + 56 kDa Composed fromComposed from leucin-repeat, leucin-repeat, zzinincc-ribbon, helix-turn-helix-ribbon, helix-turn-helix

function in tr.initiationfunction in tr.initiation Recruitment of TFIIH to PICRecruitment of TFIIH to PIC A possible role in promoter-melting through the zinc-ribbon A possible role in promoter-melting through the zinc-ribbon

coopcooperationeration with TFIIB?with TFIIB?

Regulates the activity of TFIIHRegulates the activity of TFIIH stimulstimulatesates CTD-kinase CTD-kinase stimulstimulatesates ATPase ATPase stimulstimulatesates/inhib/inhibits its helicasehelicase

Not necessary in vitro on supercoiled DNANot necessary in vitro on supercoiled DNA Role in NER (nucleotide excition repair)Role in NER (nucleotide excition repair)

XPA bindXPA binds s TFIIETFIIE TFIIE reTFIIE recruitscruits TFIIH TFIIH RepRepair complex involvementair complex involvement

34 34

56 56

TFIIHTFIIH The most complex of the GTFs – The most complex of the GTFs – 10 10 subunitssubunits The only GTF with enzymatic activity:The only GTF with enzymatic activity:

HelicaseHelicase (ATP-dep.) (ATP-dep.) ATPase (DNA-dep.)ATPase (DNA-dep.) CTD-kinaseCTD-kinase

Kinase substratKinase substratee:: CTDCTD TBPTBP TFIIETFIIE TFIIF (RAP74)TFIIF (RAP74)

TFIIHTFIIH

TFIIH-structureTFIIH-structure Multisubunit factor Multisubunit factor ( ( humanhuman / / yeastyeast ) )

89 kDa 89 kDa XPBXPB / / SSL2SSL2 (p105) NER-function ATPase/ (p105) NER-function ATPase/3´-5´-3´-5´-helicasehelicase

80 kDa 80 kDa XPDXPD / / RAD3RAD3 (p85) NER-function ATPase/ (p85) NER-function ATPase/5´-3´-helicase5´-3´-helicase 62 kDa 62 kDa P62P62 / / TFB1TFB1 (p75) UV-hypersens. (p75) UV-hypersens. 50 kDa 50 kDa P52P52 / / TFB2TFB2 (p55) (p55) 44 kDa 44 kDa P44P44 / / SSL1SSL1 (p50) (supr. of stem-loop) (p50) (supr. of stem-loop) zzininccfinger motfinger mot 34 kDa 34 kDa P34P34 / / TFB4TFB4 (p37) sinkfinger motiv (p37) sinkfinger motiv 32 kDa 32 kDa MAT1MAT1 / / TFB3TFB3 (p38) ringfinger motiv, cdk-assembly (p38) ringfinger motiv, cdk-assembly

faktorfaktor 38 kDa 38 kDa cyclin Hcyclin H / / CCL1CCL1 (p45+p47) cyclin-partner for CDK7/MO15 (p45+p47) cyclin-partner for CDK7/MO15

and Kin28and Kin28 40 kDa 40 kDa CDK7, MO15CDK7, MO15 / / KIN28KIN28 (p32) cyclin-avhengig kinase (p32) cyclin-avhengig kinase

TFIIH dual function: in transcription TFIIH dual function: in transcription initiation and in NER.initiation and in NER.

core

kinase

HoloTFIIHHoloTFIIH HoloTFIIH = [6 subenh. core] + CAK/TFIIK HoloTFIIH = [6 subenh. core] + CAK/TFIIK (cyclin-cdk)(cyclin-cdk)

TFIIH structureTFIIH structure

TFIIH multiple functionsTFIIH multiple functions Function 1: Function 1: promoter-meltingpromoter-melting assisted by helicases ( assisted by helicases (2 2

steps, see belowsteps, see below)) Model: 3´-5´-helicase + 5´-3´-helicase + ATP Model: 3´-5´-helicase + 5´-3´-helicase + ATP chain separation chain separation around TSSaround TSS

ATP-depenATP-dependendent step in initation (in addition to CTD phosphoryl.)t step in initation (in addition to CTD phosphoryl.) Function 2: Function 2: CTD-kinaseCTD-kinase, role in promoter clearance, role in promoter clearance

Model: CTD-phosphorylation after chain separation and Model: CTD-phosphorylation after chain separation and initiation initiation PIC disrupted PIC disrupted elongation complex leaves the elongation complex leaves the promoterpromoter

Function 3: role in Function 3: role in elongationelongation Model: TFIIH-kinase+ ATP Model: TFIIH-kinase+ ATP maintains hyperphosphorylated pol.II maintains hyperphosphorylated pol.II (counteracting the CTD fosfatase)(counteracting the CTD fosfatase)

Function 4: role in Function 4: role in DNA-repairDNA-repair (NER) (NER) 5 of 9 subunits of TFIIH with a double function in tr.+repair5 of 9 subunits of TFIIH with a double function in tr.+repair aacctively tr.genes are preferentially repairedtively tr.genes are preferentially repaired TFIIH can complement NER-deficient extractTFIIH can complement NER-deficient extract

Assists in formation of Assists in formation of open complex and open complex and promoter escapepromoter escape

1. ATP-dependent promoter melting - 1. ATP-dependent promoter melting - chain separation - open transcr. chain separation - open transcr. complexcomplex

2. ATP-dependent structural transition 2. ATP-dependent structural transition into an escape- competent conformationinto an escape- competent conformation

TFIIH helicase

TFIIH helicase

DNA melting helps in two DNA melting helps in two stepssteps

(A) Promoter melting. .. transcription-bubble. RNAPII = dark blue; TBP, IIB, and IIF = light blue; and TFIIH ERCC3 = open red rectangle. ATP-dependent changes are highlighted in yellow.

(B) Promoter escape. TFIIH function in promoter escape involves stimulating escape by transcription elongation complexes stalled after synthesis of 10 to 17 nt of RNA. TFIIH translocates with RNAPII during synthesis of the first 10 to 17 nt of RNA, and thus the helicase interacts with DNA downstream of the bubble. In the presence of ATP, ERCC3 rotates the DNA segment downstream facilitating extension of the transcription bubble.

TFIIH: also linked to the cell TFIIH: also linked to the cell cyclecycle

The TFIIH kinase = CAK = The TFIIH kinase = CAK = cdk7 + cyclin H + MAT-1cdk7 + cyclin H + MAT-1 CAK aCAK activates otherctivates other cdk cdks withs with Thr- Thr-phosphorylationphosphorylation

MAT-1 (ring-finger protein) MAT-1 (ring-finger protein) makesmakes CAK CAK constutive activeconstutive active (Thr-uavh) (Thr-uavh)

cdk7 alone responsible for cdk7 alone responsible for TFIIHs CDK-phosphorylationTFIIHs CDK-phosphorylation

ModelModel

Core TFIIHTFIIK/CAK

Repair-proteins

Core TFIIHTFIIK/CAK

TFIIK/CAK

Transcription DNA repair

Cell cycle

PIC assembly - a gradual PIC assembly - a gradual wrapping process?wrapping process?

TBP

TFIIB

TFIIETFIIF

RNAPIITBP

TFIIBRNAPIITBP

Topology Topology modelmodel

DNA-binding and activating functions in a transcription factor may comprise independent domains of the protein.

Coactivators - Coactivators - molecular bridges molecular bridges + chromatin remodeling+ chromatin remodeling

ChromatinRemodelling

coactivatorupstream transactivator

basal tr.app.

TFs does not affect the basal transcriptional apparatus directly,but indirectly through coactivators

3 main types of general 3 main types of general coactivatorscoactivators

1. 1. TAFsTAFs TBP-associated factors (TFIID = TBP + TAFs)TBP-associated factors (TFIID = TBP + TAFs) Multiple complexes that contain TBPMultiple complexes that contain TBP Multiple complexes that contain TAFsMultiple complexes that contain TAFs

2. 2. Mediator/SRB-complexMediator/SRB-complex ((holoenzyme holoenzyme componentscomponents))

RNAPII- associated factorsRNAPII- associated factors 3. 3. General cofactorsGeneral cofactors

Non-associated factorsNon-associated factors

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