PowerPoint PresentationTranscription in EukaryotesSlide 3Eukaryotic GenesSlide 5Slide 6Slide 7Slide 8Slide 9Slide 10Understanding Eukaryotic TranscriptionTranscription Factor ClassesSlide 13Slide 14General Transcription FactorsA Few Things to RememberRNAP II Complex DNA Binding FactsSlide 18Slide 19Assembly of the PICEach Component Contributes to Activity‘Basal’ Promoter ElementsThe TATA boxTBP Induced DNA BendingTBP Functions in Many ComplexesTFIIDSlide 27TAFs are Required at a Subset of GenesSlide 29Three Classes of TAF-Dependent PromotersTAFs are Activator SelectiveSpecialized TFIID ComplexesSlide 33TFIIATFIIA Stabilizes TFIID:DNA ComplexTFIIA and TFIIDTFIIBTFIIB Binds Along One Face of Bent DNAStable Structure Built with Multiple Weak ContactsSo Far…TFIIE & TFIIFTFIIF Is Required for InitiationTFIIF Inhibits Abortive Initiation TFIIE Helps in the Presence of TFIIHPre-Initiation Complex (PIC)TFIIHSlide 46XPB Subunit of TFIIH Melts DNASlide 48Slide 49Slide 50Initiation, Clearance and ElongationPhosphorylation of the carboxyl-terminal domain (CTD) corresponds with active transcriptionPost Release StepsThe Carboxyl Terminal Domain (CTD) of RNAP IIRepeats are Highly ConservedBasic CTD Phosphorylation EventsCTD Binding ProteinsSlide 58Post-Initiation Events1Readings for next week: Book:Molecular Biology by WeaverChapter 12Reviews:The chromatin signaling pathway: diverse mechanisms of recruitment of histone-modifying enzymes and varied biological outcomes.Smith E, Shilatifard A.Mol Cell. 2010 Dec 10;40(5):689-701.The yeast PHO5 promoter: from single locus to systems biology of a paradigm for gene regulation through chromatin.Korber P, Barbaric S.Nucleic Acids Res. 2014;42(17):10888-902.Journal Club:Genome-wide nucleosome specificity and directionality of chromatin remodelers.Yen K, Vinayachandran V, Batta K, Koerber RT, Pugh BF.Cell. 2012 Jun 22;149(7):1461-73.2Transcription in EukaryotesThe core (basal) machineryChromatin and chromatin remodeling complexesIntracellular hormone receptor mediated transcriptionOrder and dynamic recruitment3Points for the lecture:Basic principles are similar for prokaryotic and eukaryotic transcription initiation (e.g., -10 box and TATA box)Core RNAP II is composed of many subunits (TFII = transcription factor for RNAP II); increase modularityEach TFII complex contributes to either initiation and/or elongationContributions of each TFII complexes depends on promoter elementsPhosphorylation of CTD is critical for multiple transcription steps4Eukaryotic Genes5Eukaryotic RNAPs6Each RNAP is Composed of Multiple Subunits7Some Subunits Are Shared8Assembled RNAP II9RNAP II Regulation10Post-Translational/Assembly Modifications Are Common11Understanding Eukaryotic TranscriptionTAFs12Transcription Factor ClassesGeneral Transcription Factors (GTFs)Lead to accurate transcription initiation by RNA Pol II in vitro.Regulators (a.k.a., Activators and Repressors)Bind sequence-specific enhancer elements located upstream of the core promoter; stimulate or suppress the rate of transcription in response to physiological or developmental stimuli.CoregulatorsEnhance or stifle the interaction between the activator/repressors and the RNA Pol II machinery.13Generic RNAP II Promoter14The Basal Machinery15General Transcription Factors16A Few Things to RememberAssemble in a specific order to form a Preinitiation Complex.DA - DAB - DABPolF - DABPolFEHJ or D - DB - DBPolF - DBPolFEHJATBPEvolutionarily conserved. / Binds to TATA box. / Binds to the minor groove of the DNA.TAFIIsInteracts with the promoter. / Binds to TBP. / Interacts with gene-specific Tnx factors. TAFII250 has enzymatic activities--HAT and Kinase activities.TFIIAMultiple subunits (2-3). / Binds to TBP. / Stabilizes TFIID binding to DNA. / Works like TAFIIs.TFIIBSingle subunit. / Weak resemblance to the sigma factor in E. coli.. / Essential for recruitment of Pol II-TFIIF complex. / Positions the polymerase at the transcription start site.TFIIFTwo subunits: RNA Pol II-asoc. prot. (RAP30, RAP70). RAP30 has weak homology to sigma in E. coli (pol II interaction domain.) Like sigma, TFIIF reduces non-specific binding. Interacts with DAB complex.TFIIETwo subunits, 56 kDa and 34 kDa---tetramer. 56 kDa subunit contains a Zinc-finger DNA-binding motif. Interacts with DABPolF complex.TFIIH 1. Has Kinase activity: Phosphorylates the Carboxy Terminal Domain (CTD) of RNA Pol II. This kinase reaction happens only when Pol II is bound to DNA.CTD Phosphorylation-- Required for Promoter clearance. Releases Pol II from TBP. Enhances RNA processing. 2. Has Helicase/ATPase activities--required for promoter clearance.TFIISAffects elongation by limiting polymerase pausing at defined sites.TFIIFAffects elongation by limiting polymerase pausing at random sites.17RNAP II Complex DNA Binding FactsConserved Promoter SequencesCell Type Location/SequenceProkaryotic -10 -35TATAAT TTGACAEukaryotic ~ -25 ~ -80TATA (TATA box) CAAT (CAAT box)As DNA unwinds during bacterial transcription, the transcription apparatus occupies different sites in the gene during different steps of transcription.Transcription Step Distance Covered SiteInitial complex 75 bp -55 to +20Start of elongation 60 bp -35 to +25During movement 30 bp 10 to +20Order Factor Protected Fragment1. TFIID -42 to -17 (binds TATA box)2. TFIIA -80 to -173. TFIIB -80 to -17 and -10 to +104. RNA Polymerase II -80 to +155. TFIIE -80 to +3018DNA Footprint Increases with Assembly19Assembly of the Basal Machinery into a Pre-Initiation Complex20Assembly of the PIC21Each Component Contributes to Activity22‘Basal’ Promoter ElementsTFIIB TBP TFIID TFIID23The TATA boxCommon in highly-expressed genes.Determines where RNA pol II binds/starts TX.24TBP Induced DNA Bending25TBP Functions in Many Complexes26TFIID27TFIID Subunits28TAFs are Required at a Subset of Genes29Certain TAFs Bind DNA at Inr30Three Classes of TAF-Dependent PromotersThe percentage of the yeast genome dependent on each TAF ranges from 3% (TAF2) to 59–61% (TAF9). Approximately 84% of yeast genes are dependent upon one or more TAFs and 16% of yeast genes are TAF independent.31TAFs are Activator Selective32Specialized TFIID Complexes3334TFIIAComposition: TFIIA consists of two subunits in yeast and three in humans and drosophila (although two subunits are derived from a precursor protein). Interactions: TFIIA binds directly to TBP and stabilizes its binding to DNA, perhaps through
View Full Document
Unlocking...