RNA polymerase #1Pathway for Gene ExpressionReaction catalyzed by RNA polymeraseSequential addition of ribonucleotidesE. coli RNA polymeraseTwo Definitions of PromoterRNA polymerase at a promoter3-D images of core and holoenzymeHoloenzyme for RNA polymerase from E. coliRole of a subunit in assembly of RNA polymerase and other functionsMode of action of s factorsEvents at initiation of transcriptionTranscription cycleDiagram of Transcription Cycle in BacteriaSites on RNA polymerase coreTranscription bubble and RNA polymeraseEffects of transcription on supercoiling of the template3 RNA polymerases in eukaryotesSubunit structure of eukaryotic RNA polymerasesSubunits of yeast RNA Pol IIDistinct forms of RNA polymerase used for initiation and elongation: RNA Pol II3-dimensional view of yeast RNA Pol IIRNA polymerase #1General propertiesE. coli RNA polymeraseEukaryotic RNA polymerasesPathway for Gene ExpressionDNApre-mRNAproteintranscriptiontranslationreplicationreverse transcriptionmRNAForm endsSplicepost-translationalmodificationsfunctional proteinReaction catalyzed by RNA polymerase•Catalyzes the synthesis of RNA directed by DNA as a template = transcription•Makes an RNA chain with a sequence complementary to the template strand of DNA•Does NOT require a primer; can start RNA synthesis at a site on the DNA templateSequential addition of ribonucleotidesONH2NNNNOCH2OPOO-OHONH2NNNNOCH2OPO-OHOOOHRNAnn-1NH2NNNNOCH2OPO-OHOHOn+1NH2NNNNOCH2OPOPOPOO-O-O-OHOH-OOO+ATPONH2NNNNOCH2OPOO-OHONH2NNNNOCH2OPO-OHOHOOHRNAnn-1POPOO-O-O--OO++Pyrophosphate = PPiE. coli RNA polymerase•Synthesizes all classes of RNA–mRNA–rRNA–tRNA•Core= 2’ catalyzes elongation of an RNA chain•Holoenzyme = 2’ catalyzes initiation or RNA synthesis specifically at a promoterTwo Definitions of Promoter•1. The sequence of DNA required for accurate, specific initiation of transcription•2. The sequence of DNA to which RNA polymerase binds to accurately initiate transcription•In most cases, RNA polymerase binds to a DNA sequence including the initiation site, but it can be directed there by sequences flanking the initiation site. Thus definition 2 can be a subset of definition 1.RNA polymerase at a promoter-35-10ββ'αασUP: assembly and binds to UP’: form catalytic center: binds -10 and -35 of promoter to confer specificityduring initiation3-D images of core and holoenzymeCore HoloenzymeHoloenzyme for RNA polymerase from E. coli Similar to DNA polymerase I KlenowChannel with probable DNA-binding and active sites. 25 Angstroms in diameter and 55 Angstroms long (enough for about 16 bp of DNA).View perpendicular to channel:"thumb""fingers"Darst, S.A., E.W. Kubalek & R.D. Kornberg (1989) Nature 340: 730-732.(view flipped 180 )oAo100thick160Ao100AoRole of  subunit in assembly of RNA polymerase and other functionsMode of action of  factors•The  factor causes RNA polymerase to be selective in its choice of initiation sites by affecting the dissociation rate of polymerase from DNA.–Core dissociates from general DNA with a half-time of 60 min; use in elongation.–Holoenzyme dissociates from general DNA with a half-time of 1 sec! –Holoenzyme dissociates from promoter DNA with a half-time of hours (in absence of rNTPs).Events at initiation of transcriptionTranscription cycle•Initiation–Holoenzyme binds to the promoter, unwinds DNA, and forms phosphodiester bonds between 7 to 12 nucleotides–Need •Elongation–  dissociates–Core elongates RNA with high processivity–May use NusA•Termination–Polymerase dissociates from template DNA and releases new RNA–Often use .Diagram of Transcription Cycle in BacteriaStart StopRNA pol coreSigmaNusARhoStartStopRNA pol holoenzymeNontemplateTemplateClosed complexOpen complexElongating complexTerminating complexSites on RNA polymerase core•Enzyme covers about 60 bp of DNA, with about 17 bp unwound = transcription bubble.•The bubble must contact the active site for polymerization.•At the beginning of the bubble, the DNA is unwound, implicating a helicase activity.•At the end of the bubble, the DNA is rewound.Transcription bubble and RNA polymeraseEffects of transcription on supercoiling of the template•The unwinding (untwisting) and rewinding of the DNA template introduces positive supercoils ahead of the polymerase and negative supercoils behind it.•Unwinding will decrease T by 1 for every 10 bp unwound. Since L=0, W=-T, and W=+1 for every 10 bp unwound.•The opposite occurs upon rewinding. T=+1 for every 10 bp rewound, and thus W=-1 for every 10 bp rewound.•In vivo, topoisomerases aid transcription.3 RNA polymerases in eukaryotesName Makes -amanitinRNA pre-rRNA insensitive Polymerase IRNA pre-mRNA very sensitive Polymerase II some snRNAsRNA pre-tRNA less sensitive Polymerase III other small RNAs some snRNAsSubunit structure of eukaryotic RNA polymerases•All 3 have multiple subunits (8 to 14 ).•MW for each polymerase is about 500,000•Some subunits are common to all 3 RNA polymerases•All 3 RNA polymerases have subunits that are homologous to the bacterial , ’ and  subunits.Subunits of yeast RNA Pol IIApproximate subunits per size (kDa) polymerase role / comment220 1 related to '130 1 related to 40 2 related to 35 < 130 2 common to all 327 1 common to all 324 < 120 1 common to all 314 210 1Distinct forms of RNA polymerase used for initiation and elongation: RNA Pol IIEukaryotic RNA polymerase IICTD of large subunit of Pol IIPol IIaCTD of large subunit of Pol IIPPPPPPPol IIokinase + ATPphosphataseModel: Phosphorylation of Pol IIa to make Pol IIo is needed to release the polymerase from the initiation complex and allow it to start elongation.CTD has repeat of (YSPTSPT)26-50.CTD = C-terminal domain3-dimensional view of yeast RNA Pol IIBoth yeast RNA Pol II and E. coli RNA polymerase core Have a similar shape and have the channel for DNA template.Images from Dr. S.