DNA transcription@生物化学精品课件.pptx

DNA Transcription,DNA Transcription,Central Dogma- “One center, two basic points”Basic features Common to DNA replication 1) Template, Unwinding and Torsion-relieving are necessary; 2) Proceed only in the 53direction; Cordycepin can prove this Uncommon to DNA replication 1) No need for primers 2) NTPs instead of dNTPs; UTP instead of dTTP 3) Lacking proof-reading activity ( error rate is 1 in 104 or 105 nts added ) 4) Specific regions (not all DNA sequence) can be transcribed 5) To a specific gene, only one strand can be transcribed Remember some nomenclature conventions,Central Dogma,Transcription,Translation,Replication,Replication,Retro-transcription,Gene expression,Coding strand, Sense strand, Crick strand,Template strand, antisense strand, Watson strand,Transcription,Translation,Bacterial RNA Polymerase,The first nucleic acid synthesizing enzyme (polynucleotide phosphorylase, PNP ) In 1955, Marianne Grunberg-Manago and Severo Ochoa reported the isolation of an enzyme that catalyzed the synthesis of RNA. For this work, Ochoa shared the 1959 Nobel Prize in Medicine with Arthur KornbergThe real E.coli RNA Polymerase In 1960, the true enzyme was identified by 4 separate groups: Sam Weiss at the University of Chicago, Jerard Hurwitz, A. Stevens and J. Bonner. This enzyme required a template, used all four rNTPs as substrates and synthesized a product with a composition similar to that of the template, and it required Mg2+.,DNA-Dependent RNA Polymerases (RNAP),Common features -DNA template: one strand is copied -Substrate NTPs (GTP, CTP, UTP, ATP) -Divalent cation (Mg2+ )Differences between DNAP and RNAP,Differences between DNAP and RNAP,1) RNAPs can initiate synthesis which involves promoter recognition.2) RNAPs can melt the DNA duplex.3) RNAPs initiation is primed by a single nucleotide, not an oligo as is the case for DNAPs.4) RNAPs make multiple contacts with the 2-OH of the incoming NTP.5) DNA scrunching occurs for RNAPs allowing abortive cycling while still retaining contact with the promoter.6) For RNAPs, the transcript is peeled away from the template; not so for DNAPs where the open cleft allows the duplex to extend out of the enzyme.7) Initiation of synthesis is regulated by many proteins for RNAPs, but not for DNAPs.8) RNAP has no proofreading activity (error rate is 1 in 104 or 105 nts added)9) RNAP incorporates NTPs instead of dNTPs0) RNAP incorporates UTP instead of dTTP,Structure and Function of the RNAP in Prokaryotes,All three classes of RNAs are transcribed by the same RNA polymerase In E.coli, RNAP is 465 kD complex, with 2 , 1 , 1 , 1 , 1 subunit HoloenzymeCore enzyme is 2 , 1 , 1 , 1 Inhibitors Rifampicin & Streptolydigin,CORE ENZYMESequence-independent,nonspecific transcriptioninitiation,+, SUBUNITinterchangeable,promoter recognition,The assembly pathway of the core enzyme,(the w subunit makes this more efficient),In the Holoenzyme: binds DNA binds NTPs and together make up the active site subunits appear to be essential for assembly and for activation of enzyme by regulatory proteins. They also bind DNA. s recognizes promoter sequences on DNA,The sigma subunit,The sigma subunit does two things: (1) It reduces the affinity of the enzyme for non-specific DNA. (2) It greatly increases the affinity of the enzyme for promoters.,E.coli also has six alternative sigma factors that are used in special circumstances,Some of the sigma factors found in Bacillus subtilis,Anti-sigma factors,The importance of anti-sigma factors has been established in recent years. These factors form complexes with their cognate sigma factor, thereby inhibiting its function. One example is FlgM, which is an anti-sigma factor for the flagellar sigma factor sF. Another example is Rsd, which is an anti-70 factor. It is not present in exponentially growing E.coli cells. However, when E.coli enters stationary phase, Rsd is synthesized and acts to block the activity of 70 thereby allowing S to associate with the core RNA polymerase and direct expression of stationary phase genes.Control of sporulation in Bacillus subtilis also involves anti-sigma factors and anti-anti-sigma factors!,RNAP core structure from T. aquaticus.,RNAP has a “crab claw” shape with a wide internal channel to bind DNA and RNA.,RNAPs in Eukaryotes,RNA polymerases I, II and III transcribe rRNA, mRNA and tRNA genes, respectively,RNAP II Inhibitor,Mushrooms of the genus Amanita make a toxic cyclic octapeptide called a amanitin (鹅膏蕈碱）,This mushroom tastes good but eating it is deadly! 6 to 24 hours after eating it violent cramps and diarrhea set in3rd day sees a false remissionBy 4th or 5th day death will occur unless a liver transplant is doneThe symptoms are due to inhibition of RNAPII and manifest mainly in liver,The chemical structure of -amanitin,RNAPs in Eukaryotes,All 3 are big, multimeric proteins (500-700 kD) All have 2 large subunits with sequences similar to and in E.coli RNAP, so catalytic site may be conserved All have subunit homologs of a in E. coli RNAPHowever, the eukaryotic RNA polymerase does not contain any subunit similar to the E. coli factor.These features are shared by RNAPs across species,RNA polymerases I, II and III have structural features in common:,The subunits of yeast RNA polymerase II,Subunit,Function,RPB1RPB2RPB3RPB4RPB5RPB6RPB7RPB8RPB9RPB10RPB11RPB12,Binds DNA (and has CTD)Binds NTPsassemblyNot essential, (stress response)Target for activatorsEfficient assemblyNot essential(stress response)Not knownHelps select start siteNot knownAssemblyNot known,E. coli Homolog,bba1wa2,Size (kD),22015045322723171413101010,(PNAS January 30, 2001 98, 892-897),RNAP core structure fromT. Aquaticus ）.,RNAP II structure fromyeast.,Comparison of RNAP structuresin karyotes and eukaryotes,Note that the overall shape of the enzyme is the same.Also the overall positions of the subunit homologs are the same.,RNA Polymerase II,Most interesting because it regulates synthesis of mRNA Yeast Pol II consists of 12 different subunits named according to size, from largest to smallest (RPB1 - RPB12) RPB1 and RPB2 are homologous to E. coli RNA polymerase and RPB1 has DNA-binding site; RPB2 binds NTP RPB1 and RPB2 together make up the active siteRPB3 and RPB11 are homologs of a1 and a2,Important feature of RPB1,Although RPB1 is very similar to E. coli in sequence, structure and position in the enzyme, there is an important difference between the two subunits RPB1 is longer at the carboxy terminal end This extension of amino acid sequence at the carboxy-terminal end of RPB1 is called the CTD, which stands for C-terminal domain. This domain is unique to the largest subunit of RNAP II.It is NOT found in the related largest subunits of RNAP I or III, or in the E. coli RNAP largest subunit,The sequence of the CTD is an unusual 7 amino-acid repeat with an “extended” structure,therefore the CTD is essential for RNAP II function the CTD has a lot of S, T amino acids which can be phosophorylated it is known that RNAP II in an initiation complex has a non-phosphorylated CTD deletion studies showed that yeast require at least 13 repeats to survive elongating RNAP II has a phosphorylated CTD,RNA Polymerase II,RPB1 has C-terminal domain (CTD) or PTSPSYS 5 of these 7 have -OH, so this is a hydrophilic and phosphorylatable site,CTD is essential and this domain may project away from the globular portion of the enzyme (up to 50 nm!) Only RNA Pol II whose CTD is NOT phosphorylated can initiate transcription,Viral RNA Polymerases,T7 RNA PolymeraseT3 RNA PolymeraseSP6 RNA Polymerase,Fig. 1 Structure of transcribing T7RNA polymerase,The T7 RNA polymerase has a structure similar to bacterial RNA polymerase and eukaryotic mitochondrial and chloroplast enzymes.,Cheetham & Steitz (1999) Structure of a transcribing T7 RNA polymerase initiation complex, Science 286: 2305-2309.,Structure of the transcribing RNA polymerase,Detailed Transcriptional Mechanism,Three-step process 1) Initiation 2) Elongation 3) TerminationDNA transcription in prokaryotesDNA transcription in eukaryotesIn vitro DNA transcription,Prokaryotic DNA Transcription,Initiation 1) what is promoter? 2) how to determine the promoter sequences?-DNase I footprinting 3) Consensus sequences 4) Formation of transcriptional complexElongationTermination,Radio-labeling,DNase I Partial Digestion,Missing segments,Electrophoresis,Autoradiography,Footprinting,Mapping Promoters - DNA sequences that guide RNAP to the start of a gene (transcription initiation site),-10 region,RNAP binds a region of DNA from -40 to +20,The sequence of the non-template strand is shown,TTGACA16-19 bp. TATAAT “-35” spacer “-10”,Properties of Promoters,Promoters typically consist of 40 bp region on the 5-side of the transcription start site Two consensus sequence elements: The -35 region, with consensus TTGACA The Pribnow box near -10, with consensus TATAAT - this region is ideal for unwinding - why? “UP element” (an AT-rich sequence about 20 bp in size located immediately upstream of the -35 region;). The seven E. coli rrn genes, which encode ribosomal RNA, are unusually strong,Important Promoter Features (tested by mutations),The closer the match to the consensus, the stronger the promoter (-10 and -35 boxes) The absolute sequence of the spacer region (between the -10 and -35 boxes) is not important The length of the spacer sequence IS important:TTGACA - spacer (16 to 19 base pairs) - TATAAT Spacers that are longer or shorter than the consensus length make weak promoters,Binding of RNAP to Template DNA,Polymerase binds nonspecifically to DNA with low affinity and migrates, looking for promoter Sigma subunit recognizes promoter sequence RNA polymerase holoenzyme and promoter form closed promoter complex (DNA not unwound) - Kd = 10-6 to 10-9 M Polymerase unwinds about 12 pairs to form open promoter complex - Kd = 10-14 M,Initiation of Transcription,Binding of RNAP to Template DNARNA polymerase has two binding sites for NTPs Initiation site prefers to binds ATP and GTP (most RNAs begin with a purine at 5-end) Elongation site binds the second incoming NTP 3-OH of first attacks alpha-P of second to form a new phosphoester bond (eliminating PPi) When 6-10 unit oligonucleotide has been made, sigma subunit dissociates, completing initiation,Transcriptional regulation in bacteria,2. Interaction of RNA polymerase with template,s,s,Sigma leavesafter 10 nt aretranscribed.,DNA wrapping model (B. Coulombe),Sigma joinscomplex,When does sigma leave?,Is sigma still present after fingersclose, but before abortive cyclinghas stopped?,?,Finding and binding the promoter,Transcriptional regulation in bacteria,Transcription cycle,Fig. 9.9,Abortive initiation or cycling:RNA pol transcribe 2-9 nt andthen restarts. Does not leavethe promoter. May occur severalhundred times before true elongation.Note: the number of bases that canbe packed into the active site of the enzyme is 8. This correlates withabortive products of 8-10 bases.,Transcriptional regulation in bacteria,Fig. 9.9,Sometimes RNA polymerase pausesdue to a temporary shortage of thecomplementary nucleotide. When thisoccurs, restarting synthesis requiresthe GreA and GreB proteins to releasethe pause. The 3 end is cleaved so that it is properly aligned within thecatalytic site of the polymerase again.,Chain Elongation,Core polymerase - no sigma factorPolymerase is pretty accurate - only about 1 error in 10,000 bases (not as accurate as DNAP III)Even this error rate is OK, since many transcripts are made from each gene Elongation rate is 20-50 bases per second - slower in G/C-rich regions and faster elsewhere Topoisomerases precede and follow polymerase to relieve super coiling,Science, vol. 281, p 424 (1998),Spatial Organization of Transcription Elongation Complex in E. coli,Interactions between nucleic acids and the core enzyme keep RNAP processive,Two mechanisms Rho() - the termination factor protein rho is an ATP-dependent helicase it moves along RNA transcript, finds the bubble, unwinds it and releases RNA chain Specific sequences - termination sites in DNA inverted repeat, rich in G:C, which forms a stem-loop in RNA transcript 6-8 As in DNA coding for Us in transcript,Chain Termination,Rho-independent transcription termination (depends on DNA sequence - NOT a protein factor),Stem-loop structure,Rho-independent transcription termination,RNAP pauses when it reaches a termination site. The pause may give the hairpin structure time to fold The fold disrupts important interactions between the RNAP and its RNA product The U-rich RNA can dissociate from the template The complex is now disrupted and elongation is terminated,Rho-Dependent Transcription Termination(depends on a protein AND a DNA sequence),G/C -rich site,RNAP slows down,Rho helicase catches up,Elongating complex is disrupted,Differences Transcription Bacteria vs. Eukaryotes,Multiple Polymerases at least 3 types of RNAPsChromatin and NucleosomesUnable to initiate transcription on their own - Require Transcription Factors (TF)Unable to recognize Promoters on their own Primary transcripts contain exons The Promoters are complex. Multiple regulatory proteins can bind to the promoter. Cis-acting elements and Trans-acting factors.Enhancer, silencer & insulatormRNAs are mostly monocistronic Genes controlled by positive control - off unless activators are present In eukaryotes, transcription and translation occur in separate compartments.,Enhancers can occur in a variety of positions with respect to genes,Transcription unit,P,Enhancer,Enhancer,Adjacent,Downstream,Internal,Distal,Upstream,Trans-acting factor,Transcription factor,Other Cis-acting Elements,ERE - Estrogen response element HSE - Heat shock element MRE - Metal response element GRE - Glucocorticoid response element,DNA transcription by RNAP I,Promoters 1) Core promoter (-45 to +20) 2) Upstream control element (UCE; -180 to -107) Distance between UCE and promoter is critical Species-specific TFs 1) SL1: TBF and TAF 2) UBF Mechanism (1) Initiation & Elongation (2) termination-requires a specific DNA-binding termination factor,UCE,Core Promoter,Start-point,DNA transcription by RNAP III,Promoters 1) Some Pol III genes (tRNA, 5S rRNA) have internal promoters 5S rRNA: Box A & Box C tRNA: Box A & Box B 2) Some are “pol II-like” Eg: some snRNAs have essential TATA box and upstream promoter elementsTFIII TFIIIA、B and C: TFIIIC binds both A box and B box. TFIIIB: TBP、BRF（TFIIIB-related factor， and TFIIIBMechamism (1) Initiation & Elongation (2) Termination-terminates after U-series, but no apparent upstream stem-loop,A Box,B Box,Startpoint,DNA transcription by RNAP II,Promoters-usually contain one or more of the following: Initiator ,TATA box (Hogness box） and Upstream element（UPE) 1) Initiator (Inr): consensus sequence PyPyANT/APyPy; (A is +1) 2) TATA box Consensus sequence TATAAAA 3) Upstream elements GC boxes (GGGCGG)-Binding site for Sp1 CCAAT box -Binding site for CCAAT- binding transcription factor (CTF) and CCAAT/enha