Basal Transcription Regulation of Transcription Lecture 4 Basics of Regula on 2 general levels of gene regula on 1 transcrip on RNA synthesis 2 transla on protein produc on genome transcriptome proteome total RNA produced can control what s transcribed protein components can control which proteins are made gene regula on transcrip on transla on genome transcriptome proteome Transcrip on Basics of Regula on Euks Have 3 RNA Pols RNA pol I RNA pol II RNA pol III transcribes genes that encode proteins all proteinGcoding genes plus snoRNA genes miRNA genes siRNA genes most snRNA genes RNA pol II transcribes proteinGencoding genes Transcrip on RNA Pol II RNA POL II Subunit Composi on best characterized enzyme from a yeast has 12 subunits 1 220 kDa subunit binds DNA CTD CGTerminal Domain consists of 26 tandem repeats of YSPTSPS TyrGSerGProGThrGSerGProGSer heptad sequence CGterminus tail must be phosphorylated before it can be transcribed 2 150 kDa subunit 1 copy 3 45 kDa subunit 2 copies 4 some subunits common to all pols 3 all 40 kDa in size 12 subunits 220 kDa w YGSPGTGSP CTD 150 kDa 45 kDa 3 small ones Transcrip on RNA Pol II Terminology upstream 5 to point of reference downstream 3 to point of reference transcrip on 1 strand of DNA copied into RNA start site nucleo de which RNA synthesis begins transcrip on starts 1 site ELEMENTS DNA FACTORS PROTEIN upstream downstream transcrip on start site 1 site Transcrip on Terms PROMOTERS promoter necessary su cient for ini a on of accurate transcrip on speci es chromosome regions transcribed together promoter DNA sequence necessary su cient for ini a on of accurate transcrip on speci es regions of chromosomes transcribed together as a transcrip on unit a chromosome is NOT copied into 1 transcrip on unit promoter ini ates accurate transcrip on of 1 transcrip on unit Transcrip on Promoters modular has sets of sequence blocks Core Promoter Elements involved in specifying 1 site loca on sequences found in many promoters used cons tu vely not regulated PromoterQProximal Elements control transcrip on frequency shared by many0but0not0all genes found within 200 bases of start site modular organiza on allows di erent combos of core proximal elements0 Distal Elements farther from promoter control transcrip on frequency speci c promoters e g enhancers RNA POL II PROMOTERS Design modular organiza on di combos of core proximal elements0 core promoter elements cons tu ve common promoterQproximal elements shared by many genes distal elements control transcrip on freq speci c promoters modular organiza on core promoter elements 1 site promoterGproximal elements distal elements0 Transcrip on Promoters core promoter shortest sequence of DNA that directs transcrip on modular has 1 or more of 4 sequence blocks 1 2 ini ator Inr consensus Py 2CA Py 5 located G3 to 5 remember start site is 1 usually an A nucleo de surrounded by pyrimidines Py recognized by TFIID Transcrip on Factor IID other proteins TATA box 7Gbp consensus TATAAAA binding site for TBP TATAGBinding Protein located G25 to G35 3 BRE Transcrip on Factor IIB TFIIB GRecogni on Element recognized by TFIIB 4 DPE Downstream Promoter Element CORE PROMOTER ELEMENTS shortest transcrip onGdirec ng modular 1 or more of sequence Inr G3 to 5 Py 2CA Py 5 recognized by TFIID TATA box G25 to G35 TATAAAA TBP binding site recognized by TFIIB BRE DPE shortest sequence modular 1 or more of Inr TATA box BRE DPE Transcrip on RNA Pol II Core Promoter Elements0 PROXIMAL UPSTREAM SEQUENCES Elements 3 kinds 1 octamer 2 CAAT box 3 GCQbox upstream of TATA box same sequence can be present in di erent 3 kinds octamer CAAT box GCQbox promoters levels can s mulate transcrip on beyond basal upstream of TATA box same one can be in various promoters s mulate transcrip on can change freq of transcrip on but cannot change transcrip on start site loca on 3 kinds octamer CAAT box GCGbox s mulate transcrip on Transcrip on RNA Pol II Proximal Sequence Elements TRANSCRIPTION FACTORS TFs RNA pol II enzyme holoenzyme synthesizes RNA cannot recognize CORRECT sites to begin transcrip on proper es of TFs work w RNA pol II but are not a direct part of RNA pol II determine speci city of transcrip on speci city correct start site selec on on chromosome a ect e ciency of promoters e ciency amount of RNA synthesized per unit me RNA pol II makes RNA can t see 1 site TF proper es speci city e ciency Transcrip on Transcrip on Factors Classi ca on of TFs GTFs General Transcrip on Factors determine start site required for ini a on of transcrip on from promoters nomenclature TFIIX where X A B C etc upstream factors GTFs pick start site upstream factors Transcrip on Transcrip on Factors Classi ca on very important in loca on orienta on control of start site contains TBP TATAGBinding Protein bound to TATA box TBP has TAFs TBPGAssociated Factors TFIID complex TFIID TFIID makes mul ple contacts w core promoter over 50 of promoters lack TATA box TFIID TBP rst to arrive on scene recruit RNA pol II other proteins to start site picks start site loca on orienta on contains TBP bound to TATA box TBP has TAFs complex TFIID TFIID makes contacts w core promoter TBP bound to TATA box TBP has TAFs complex TFIID makes contacts w core promoter Transcrip on TFIID INITIATION COMPLEX TFIIABF Assembly TFIIA A binds recruits B binds TFIIDGpromoter complex TFIIF increases TBP s a nity for TATA box 10Gfold in0vitro binds TAF TFIIB bridging protein that brings RNA pol II to promoter binds RNA pol II binds TFIIDGpromoter complex helps orient RNA pol II to select start site TFIIF RAP30 subunit ghtly binds RNA pol II important for recruitment of RNA pol II to preQini a on complex has ATPGdependent DNA helicase in RAP70 subunit unwinds duplex DNA to form an OPEN complex TFIIA TFIIB TFIIF Transcrip on Assembly of Ini a on Complex TFIIABF BASAL TRANSCRIPTION COMPLEX Assembly TFIIF helicase other factors CTD heptad repeat YSPTSPS 25G52 copies CGterminus TFIIE binds RNA pol II recruits binds TFIIH TFIIH has helicase kinase RNA pol II TFIIE TFIIF TFIIH CTD heptad repeat YSPTSPS Transcrip on Assembly of Basal Transcrip on Complex TFIIEFH PROMOTER CLEARANCE TFIIEH TFIIH TFIIE needed for postGini a on promoter clearance TFIIH TFIIE E binds recruits H binds RNA pol II recruits TFIIH into open complex ATP required TFIIH TFIIE facilitate shin from ini a on elonga on stage of transcrip on by RNA pol II XPB subunit DNA helicase Cdk7 kinase subunit phosphorylates CTD of RNA pol II on serines marks