General Transcription Initiation FactorsAssay for accurate initiation by bacterial RNA polymeraseRandom transcription by bacterial RNA polymerase coreRandom transcription by eukaryotic RNA polymeraseAccurate initiation by Euk RNA polymerase II plus factors in the nucleusGeneral transcription factors = GTFsFractionation of nuclear extracts to find GTFs Phosphocellulose column: ion exchangeFractionation of nuclear extracts to find GTFs Fractionation scheme, DEAE celluloseGTIFs for RNA polymerase IITATA Binding Protein = TBPTBP bound to DNARNA Pol II bound to DNA and general transcription initiation factorsMovie of TBP, then binding to TATA DNA (and bending it), and then TFIIA binding to TBP-TATASequential Binding Model for assembly of preinitiation complexDirect Binding of Holoenzyme model for assembly of the preinitiation complexOther proteins involved in transcription and regulationRNA polymerase II Holoenzyme and MediatorExpanding the functions of RNA polymeraseParallels between initiation pathway in prokaryotes and eukaryotesGTFs for RNA polymerase IGTFs for RNA polymerase IIIGeneral Transcription Initiation FactorsAssay for accurate initiation by bacterial RNA polymeraseβαασPromoterE. coli RNA polholoenzyme+βαασinitiationelongationNascent RNARun-off transcript,Discrete size+ +ββ'ααββ'ααRNA Pol coreRandom transcription by bacterial RNA polymerase corePromoterE. coli RNA pol core+Random initiationelongationTranscripts of random sizeββ'ααββ'ααββ'ααββ'ααββ'ααββ'ααRandom transcription by eukaryotic RNA polymerasePromoterEukaryotic RNA pol II+Random initiationelongationTranscripts of random sizeAccurate initiation by Euk RNA polymerase II plusfactors in the nucleusPromoter+initiationelongationNascent RNARun-off transcript,Discrete size+ +Eukaryotic RNA pol II+Nuclear extract, S-100GeneralTranscription InitiationFactorsGeneral 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 exchangeTemplate: Adenovirus late promoterPlusPurifiedPol IIRun-offTranscript:AccurateInitiation at promoterPolyacrylamide gel separating products of in vitro transcriptionFractionation of nuclear extracts to find GTFsFractionation scheme, DEAE celluloseMatsui, Segall, Weil, Roeder (1980) JBC 255:11992PlusPurifiedPol IIRun-offTranscriptAccurateInitiation at promoterGTIFs for RNA polymerase II TFIIDTBP}TAFsIIBIIAIIEIIFIIHhelicaseprotein kinaseTBPInrIIBIIAPol IIaIIFIIEIIHCTD of large subunit of Pol IIRecognize core promoterTargets Pol II to promoterModulates helicaseHelicaseCTD protein kinaseMany 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 DNAImage from crystal structures, provided by Dr. T. Nixon.RNA Pol II bound to DNA and general transcription initiation factorsRNA polymerase II from yeast(does not include subunits 4 and 7)136Ao140AoAo100thickChannel 25Aodiameter Groove 25Aowideflexible end of "thumb"Channel plus groove could accomodate 20-25 bp of duplex DNATFIIADNATBPTFIIEDarst, 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 resolutionAoTFIIBMovie of TBP, then binding to TATA DNA (and bending it), and then TFIIA binding to TBP-TATAThanks to Drs. Song Tan and Tracy Nixon for moviesSequential Binding Model for assembly of preinitiation complex-30 +1TATAInrPolymerization 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.IIBEukaryotic RNA polymerase IITFIID}TBPTAFsIIBIIECTD of large subunit of Pol IIPol IIaor TBPIIAIIFhelicaseprotein kinaseIIHTATAInrIIAPol IIaIIFIIEpreinitiation complexTATAInrIIAIIBPol IIaIIFIIEATP hydrolysisinitiation complex, DNA melted at InrIIHIIH= PICActivated PICDirect Binding of Holoenzyme model for assembly of the preinitiation complexTFIID}TBPTAFs-30+1TATAInrTBPpreinitiation complexATP hydrolysisinitiation complex, DNA melted at InrorTATAInrIIBPol IIaIIFIIHIIESRB IIBTATAInrHoloenzymePol IIaIIFIIHIIEIIASRB activator?IIBPol IIaIIFIIHIIESRB IIATATAIIA?= PICActivated PICOther 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 functionRNA 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?) = HoloenzymeExpanding the functions of RNA polymerasePolymeraseTrans-cribeStart atPromoterRespond toActivatorRNA Pol IIYesNoNoRNA Pol II +GTIFsYesYesNoRNA Pol IIholoenzyme+ GTIFsYesYesYesParallels between initiation pathway in prokaryotes and eukaryotesFrom Eick et al. (1994)Trends in Genetics 10: 292-296GTFs for RNA polymerase IEukaryotic RNA polymerase IUBF1SL1}TBPPol IGTFs for RNA polymerase IIIIIIAIIICIIIC}TFIIIBTBPPol IIIEukaryotic RNA polymerase