1、*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)L
2、acking 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 conventionsCentral DogmaCentral DogmaTranscriptionTranslationReplicationReplicationRetro
3、-transcriptionGene expressionCoding strand,Sense strand,Crick strandTemplate strand,antisense strand,Watson strandTranscriptionTranslation*The first nucleic acid synthesizing enzyme(polynucleotide phosphorylase,PNP)In 1955,Marianne Grunberg-Manago and Severo Ochoa reported the isolation of an enzyme
4、 that catalyzed the synthesis of RNA.For this work,Ochoa shared the 1959 Nobel Prize in Medicine with Arthur Kornberg*The 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 re
5、quired 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+.*Common features -DNA template:one strand is copied -Substrate NTPs(GTP,CTP,UTP,ATP)-Divalent cation(Mg2+)*Differences between DNAP and RNAP Differenc
6、es between DNAP and RNAPDifferences between DNAP and RNAP1)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 in
7、coming 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 regulate
8、d 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 dTTPC All three classes of RNAs are transcribed by the same RNA polymerase In E.coli,RNAP is 465 kD c
9、omplex,with 2,1,1,1,1 subunit*Holoenzyme*Core enzyme is 2,1,1,1*Inhibitors Rifampicin&Streptolydigin 2 2 2 2 2 2=core enzyme12CORE ENZYMESequence-independent,nonspecific transcriptioninitiation+vegetative(principal )7070heat shock(for emergencies)3232nitrogen starvation(for emergencies)6060 SUBUNITi
10、nterchangeable,promoter recognitionThe assembly pathway of the core enzyme(the w w subunit makes this more efficient)12 7070RNAP HOLOENZYME-70 Promoter-specific transcription initiationIn the Holoenzyme:*binds DNA*binds NTPs*and together make up the active site*subunits appear to be essential for as
11、sembly and for activation of enzyme by regulatory proteins.They also bind DNA.*recognizes promoter sequences on DNAThe sigma subunitThe sigma subunit CThe 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 fo
12、r promoters.E.coli also has six alternative sigma factors that are used in special circumstances Some of the sigma factors found in Bacillussubtilis Anti-sigma factorsAnti-sigma factorsCThe importance of anti-sigma factors has been established in recent years.These factors form complexes with their
13、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 synthe
14、sized 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.CControl of sporulation in Bacillus subtilis also involves anti-sigma factors and anti-anti-sigma factors!RNAPcorestructurefromT.aquaticus.RNAP has
15、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）鹅膏蕈碱）lThis mushroom tastes good
16、 but eating it is deadly!l 6 to 24 hours after eating it violent cramps and diarrhea set inl3rd day sees a false remissionlBy 4th or 5th day death will occur unless a liver transplant is donelThe symptoms are due to inhibition of RNAPII and manifest mainly in liverThe chemical structure of-amanitin*
17、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 in E.coli RNAP*However,the eukaryotic RNA polymerase does not contain any subunit similar to the E.coli factor.*These f
18、eatures are shared by RNAPs across speciesRNA polymerases I,II and III have structural features in common:The subunits of yeast RNA polymerase IISubunitFunctionRPB1RPB2RPB3RPB4RPB5RPB6RPB7RPB8RPB9RPB10RPB11RPB12Binds DNA(and has CTD)Binds NTPsassemblyNot essential,(stress response)Target for activat
19、orsEfficient assemblyNot essential(stress response)Not knownHelps select start siteNot knownAssemblyNot knownE.coli Homolog 11w w22Size(kD)22015045322723171413101010(PNAS January 30,2001 98,892-897)RNAP core structure fromT.Aquaticus）.RNAP II structure fromyeast.Comparison of RNAP structuresin karyo
20、tes and eukaryotesNote that the overall shape of the enzyme is the same.Also the overall positions of the subunit homologs are the same.MostinterestingbecauseitregulatessynthesisofmRNA*Yeast Pol II consists of 12 different subunits named according to size,from largest to smallest(RPB1-RPB12)*RPB1 an
21、d 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 site*RPB3 and RPB11 are homologs of a1 and a2Important feature of RPB1*Although RPB1 is very similar to E.coli in sequence,structure and position in the enzyme,there
22、 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
23、the related largest subunits of RNAP I or III,or in the E.coli RNAP largest subunitThe sequence of the CTD is an unusual 7 amino-acid repeat with an“extended”structure2652l therefore the CTD is essential for RNAP II functionl the CTD has a lot of S,T amino acids which can be phosophorylatedl it is k
24、nown that RNAP II in an initiation complex has a non-phosphorylated CTDl deletion studies showed that yeast require at least 13 repeats to survivel elongating RNAP II has a phosphorylated CTDRNA Polymerase II*RPB1 has C-terminal domain(CTD)or PTSPSYS 5 of these 7 have-OH,so this is a hydrophilic and
25、 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 PolymerasesViral RNA Polymerases*T7 RNA Polymerase*T3 RNA Polymerase*SP6 RNA PolymeraseF
26、ig.1 Structure of transcribing T7RNA polymeraseThe 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 po
27、lymeraseDetailed Transcriptional MechanismDetailed Transcriptional Mechanism*Three-step process 1)Initiation 2)Elongation 3)Termination*DNA transcription in prokaryotes*DNA transcription in eukaryotes*In vitro DNA transcription*Initiation 1)what is promoter?2)how to determine the promoter sequences?
28、-DNase I footprinting 3)Consensus sequences 4)Formation of transcriptional complex*Elongation*TerminationRadio-labeling DNase I Partial DigestionMissing segmentsElectrophoresisAutoradiographyFootprinting-10 regionRNAP binds a region of DNA from-40 to+20The sequence of the non-template strand is show
29、nTTGACA16-19 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 unwind
30、ing-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(-1
31、0 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*Polymerase binds nonspeci
32、fically 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 *Binding of
33、RNAP to Template DNA*RNA 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 uni
34、t oligonucleotide has been made,sigma subunit dissociates,completing initiation Transcriptional regulation in bacteria2.Interaction of RNA polymerase with template Sigma leavesafter 10 nt aretranscribed.Sigma joinscomplexWhen does sigma leave?Is sigma still present after fingersclose,but before abor
35、tive cyclinghas stopped?Finding and binding the promoterClosed complex formationRNAP bound-40 to+20Open complex formationRNAP unwinds from-10 to+2Binding of 1st NTPRequires high purine NTPAddition of next NTPsRequires lower NTPsDissociation of sigmaAfter RNA chain is 6-10 NTPs longTranscriptional re
36、gulation in bacteriaTranscription cycleFig.9.9Abortive 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
37、withabortive products of 8-10 bases.Transcriptional regulation in bacteriaFig.9.9Sometimes due to a temporary shortage of thecomplementary nucleotide.When thisoccurs,restarting synthesis requiresthe and proteins to releasethe pause.The so that it is properly aligned within thecatalytic site of the p
38、olymerase again.Core polymerase-no sigma factor*Polymerase 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 else
39、where*Topoisomerases precede and follow polymerase to relieve super coiling Science,vol.281,p 424(1998)Spatial Organization of Transcription Elongation Complex in E.coliInteractions between nucleic acids and the core enzyme keep RNAP processiveTwo mechanisms C Rho()-the termination factor protein rh
40、o is an ATP-dependent helicase it moves along RNA transcript,finds the bubble,unwinds it and releases RNA chain C 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-independ
41、ent transcription termination(depends on DNA sequence-NOT a protein factor)Stem-loop structureRho-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 i
42、ts RNA product The U-rich RNA can dissociate from the template The complex is now disrupted and elongation is terminatedRho-Dependent Transcription Termination(depends on a protein AND a DNA sequence)G/C-rich siteRNAP slows downRho helicase catches upElongating complex is disrupted*Multiple Polymera
43、ses at least 3 types of RNAPs*Chromatin and Nucleosomes*Unable 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-a
44、cting elements and Trans-acting factors.*Enhancer,silencer&insulator*mRNAs 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 res
45、pect to genesTranscription unitPEx1Ex2EnhancerEnhancerAdjacentDownstreamInternalDistalUpstreamTrans-acting factorTranscription factor*ERE-Estrogen response element*HSE-Heat shock element*MRE-Metal response element*GRE-Glucocorticoid response element DNA transcription by RNAP IDNA transcription by RN
46、AP 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 factorUCECore PromoterStart-poin
47、tDNA transcription by RNAP IIIDNA 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 elements*TFIII TFIIIA、B and C:TFIIIC binds both A b
48、ox and B box.TFIIIB:TBP、BRF（TFIIIB-related factor，and TFIIIB*Mechamism (1)Initiation&Elongation (2)Termination-terminates after U-series,but no apparent upstream stem-loopA BoxB BoxStartpoint*Promoters-usually contain one or more of the following:Initiator,TATA box(Hogness box）and Upstream element（U
49、PE)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/enhancer binding protein(C/EBP)*TF IIEnkaryotic Promoter*The core p
50、romoter located within about 30 bp of the start site*An upstream promoter,which may extend over as many as 200 bp farther upstream*The rows of lock boxes in a bank provide a useful analogy.*To open any particular box in the room requires two keys:*Your key,whose pattern of notches fits only the lock
