期末復習-sxl分子生物學上

MolecularBiology:thestudyofgeneandtheiractivitiesatthemolecularlevel,itgrewoutofthedisciplinesofGeneticsandBiochemistryMendel’slawsofEverycellcontainedpairsoffactorsandthateachpairdeterminedaspecifictrait(特點Themembersofeachpairsegregatedfromeachotherintheprocessofsex-cellformation,sothatagametecontainedonememberofeachpair.LawofindependentThesegregationofeachpairwasindependentofthesegregationofotherpairsofGeneisinMorganexplainedtheseparationofcertaininheritedcharacteristicsthatareusuallylinkedascausedbythebreakingofchromosomessometimesduringtheprocessofcelldivision.EachgeneistranslatedintoanenzymetoperformtaskswithinanDNAasgeneticmaterial－DNAstructure－Restriction binantDNAGene&Innon-molecularterms,geneisaunitofinheritancethaternsthecharacterofaparticulartrait.Inmolecularterms,asegmentofDNAcontainingtheinformationforasinglepolypeptideorRNAmolecule,includingtranscribedbutnon-codingregions.Ageneticunitdefinedbyacis-transtest.Forallpracticalpurpose,itissynonymouswiththewordChemicalnatureof Polynucleotides多聚核苷酸containsdeoxy)ribosephosphoricacidbasesAndbasesinclude:(ThetwostrandsofDNAmoleculearedoublehelixofantiparallelstrands,heldtogetherbyHydrogenbondformation(Guanine–Cytosine,Adenine–Thymine).Basesstackinsidethehelixtoexcludeswaterandstabilizethemselves.OneBASEPAIR=0.34nm,completeturnofhelix=3.4nm.AndeachturncontainsaMajorandMinorForsinglestrandofDNA,thereexistsdirectionorpolarity5’to3’.Andthebackbonecomprisesofchargedphosphateresidues.Sugarsarelinkedbyaphosphodiesterbond,and5’methylgroupislinkedtothe3’OHgroup.Har&JonesRNAdiffersfromRNAhasasugarribose,whenDNAhasasugarRNAcontainsuracil(U),whenDNAhasthymineRNAmoleculeismuchsmallerthanCertainescontaingenesmadeofRNAinsteadofGenomeisthecomplementofgeneticinformationuniquetoeachspeciesoforganism,whichequivalenttotheDNAofahaploidsetofchromosomesfromthatspecies. pleyseparatedeachOncethetwoDNAstrandshaveseparated,thehydrophobicinteractionsthatresultfromstackingaregreatlydecreased,whichchangestheelectronicnatureofthebasesandincreasestheirUVabsorbance(260nm).Tmisthetemperatureatwhichtheshiftinabsorbanceishalfcompleted.MoreGC,higherTm.GenomesfromdifferentSpecieshavedifferentGCcontent.DNArenaturation:whenweslowlycooladenaturedDNAsolution,DNAregainedthepropertiesofthedoublehelix.(Reassociation:DNAisshearedintopiecesofafewhundredbp,heatedtodenatureintosinglestrands,thenallowedtorenatureduringcooling.Cot1/2:thevaluewhen50%renaturationhasoccurredwhichcanbeusedtoestimatethelengthofuniqueDNAinasample.Co:theoriginalconcentrationofdenaturedAhighconcentrationofDNAincubatedforashortertime=alowconcentrationofDNAincubatedforalongertimeHigherCot1/2valuesindicategreatergenomeCvalueSizeofhaploidgenome=C-C-valueparadox:umC-valuessuggestthatsomelessevolvedorganismsmaybemorecomplexthanmoreevolvedorganismsC-valueparadoxisexplainedbyrepetitiveNon-nuclearThemitochondria線粒體andchloroplasts(葉綠體alsohaveaDNAgenomeorchromosome).Theseresembleprocaryoticgenomes(likelyduetotheendosymbioticoriginoftheseorganelles)butaremuchsmaller.MethodsofMolecularClones:agroupofidenticalcellsorGenecloning:ProducemanyidenticalcopiesofaUsesofgeneclones:TogetenoughamountofageneforfurtherysisGOI:Geneofinterest(目的)PrincipleofgeneInVitro(生物體外):PCRallowssufficientamplificationofDNAsequencestoenabledirectsequencingoruseoftheamplifiedDNAinspecificprotocolswithoutInVivo(生物體內):ProducelargetiesofthesegenesinbacteriabylinkingeukaryoticgenestosmallbacterialorphageDNAsandinsertingthese moleculesintobacterialhosts,makesuretheforeigngenecanreplicate.PCRPolymeraseChain PurposeofaPCR:tomakeahugenumberofcopiesofaThereare3majorstepsinaPCR,whicharerepeatedfor30or40cycles.Thatisdoneonanautomatedcycler,whichcanheatandcoolthetubeswiththereactionmixtureinaveryshorttime.Denaturationat94oC:doublestrandopenstosinglestrandedAnnealingat50-60oC:primersbindtotheDNAExtensionat72oC:Thebases(complementarytothetemplate)arecoupledtotheprimeronthe3'side(thepolymeraseaddsdNTP'sfrom5'to3',readingthetemplatefrom3'to5'side,basesareaddedcomplementarytothetemplate)RequirementsforaSequenceinformationoftheGOIfordesigningprimersAppropriatetemplate(DNA)Mg2+Buffer(pHandsaltdNTP(materialsformakenewDNARestrictionEndonucleasesTheenzymespreventinvasionofforeignDNA(restrictviralDNA)CharacteristicsofRestrictionEndonucleases(RE):REsrecognizespecificnucleotidesequencesandcleavebothstrandsoftheDNAcontainingthosesequences.Recognitionsequencesformanyenzymesarethesameonbothstrands.Suchrecognitionsequencesaresaidtobepalindromic(回文序列）.CommonlyusedREsalwayscleavetheDNAstrandsatafixedpositionrelativetotherecognitionsequence.Productsofcleavage:flushendsor5'or3'Isoschizomers(同裂酶):enzymesoriginatedfromdifferentorganismandrecognizeidenticalsequence.Theycouldcutthesequenceatsameordifferentsites,Isocaudomers(同尾酶someREproducecompatibaleoverhamgs,eventhoughtheirrecongnitionsequencesarenotidentical.Toavoiddestroyingthehostcell’sownDNA,restrictionendonucleasesarepairedwithmethylasethatrecognizeandmethylatethesameDNAsites.Thetwoenzymes,therestrictionendonucleaseandthemethylases,arecalledanR-M dam:adeninemethylase,methylatestheN6positionoftheadeniniedcm:cytosinemethylase,methylatestheC5positionoftheinternalcytosineCpG:methylatestheC5positionofthecytosineresidueinthedinucleitiderecognitionsequence.CpNpG:methylatestheC5positionofthecytosineresidueinthetrinucleitide.Methylation-sensitiveRE:REcannotcutifcorrespondingmethylationhappenedHostStains:labeleditsmethylationcharacteristicsDpnI:cutonlymethylatedbinantDNAisDNAthathasbeencreatedartificially.DNAfromtwoormoresourcesisincorporatedintoasingle binantmolecule.Stepsofmaking binantCut,ligate,transferintoahostVectors（載體）:carrierswhichallowreplicationof binantDNAEssentialcomponentsofavector:ORI(子):thesitewhereDNAreplicationbegins,toensurereplicationofforeignDNAAntibioticmarkergene:toselectthehostcellswiththevectorMCSMultiplecloningsite,多克隆位點siteallowingtheinsertionofforeignTypesofPlasmidsareextra-chromosomalDNAelements.Phagescontainingsingle-strandedDNAArtificialchromosomescontainalltheelementsthataDNAneedstofunctionasachromosomeinthehostorganism.YAC:yeastartificialchromosomesBAC:bacterialartificialchromosomes(Plasmidsarecircular,double-strandedDNAmoleculesthatexistinbacteriaandinthenucleiofsomeeukaryoticcells.CharacteristicsofPlasmid:Small+Usuallycarryonlyoneorafewgenes+CircularHaveasingleoriginofreplication,canreplicateindependentlyPlasmidsarereplicatedbythesamemachinerythatreplicatesthebacterialchromosome.PlasmidDNAreplicationrate>thatofthehost-multipleplasmids/cellPlasmidDNAreplicationrate<thatofthehost-singleplasmids/cellPlasmidsenterthebacterialcellwithrelativeease. PhageisanaturalvectorsystemthattransducesbacterialDNAfromonecelltoAdvantagesovertheinfectionstohostcellsaremuchmoremodate(提供muchmoreforeignDNAcanbeusedtomakeCosmids:engineeredphageswhichcan modateupto50kbinserts.Itcontainsthecossites(cohesiveends)ofλphageDNA,whichallowtheDNAtobepackagedintoλphageheads.CosmidsalsocontaintheORIofaplasmid,andbehavebothasplasmidsandItdoesnotreplicateasphage,butasplasmid(containsaplasmidORI).ItisonlyinfectiousasYAC:AvectorusedtocloneDNAfragments(upto400kb).Itisconstructedfromthe centromeric,andreplicationoriginsequencesneededforreplicationinyeastcells.:The omerewhichislocatedateachchromosomeend,protectsthelinearDNAfromdegradationbynucleases.CEN:Thecentromere(著絲粒)whichisthe entsiteformitoticspindle（紡綞體"pulls"onecopyofeachduplicatedchromosomeintoeachnewdaughterARS(autonomousreplicatingsequence):specificDNAsequencesthatallowtheDNAreplicationmachinerytoassembleontheDNAandmoveatthereplicationforks.Centromers(CEN),omeres()andautonomousreplicatingsequence(ARS)，selectivemarkersforidentifyingcellscontainingtheYACvector,andrecognitionsitesofrestrictionThetargetDNAispartiallydigestedbyEcoRIandtheYACvectoriscleavedbyEcoRIandLigatethecleavedvectorsegmentswithadigestedDNAfragmenttoformanartificialTransformyeastcellstomakealargenumberofBAC:AvectorusedtocloneDNAfragmentsof100to300kbinsertsizeinE.colicells.ItisbasedonthenaturallyoccurringF-factorplasmidfoundinE.coli.DNAlibrary:acollectionofclonedDNAfragments,includingcDNAlibraryandgenomiclibrary.cDNA:complementaryDNA,aDNAcopyofRNAcDNAlibrary:asetofclonesrepresentingasmanyaspossibleofthemRNAsinagivencelltypeatagiventime(temporalandspatialspecific)Reversetranscriptase:RNA-dependentDNApolymeraseRT-PCR:ReversetranscriptasePCRMakingacDNAUsingoligo(dT)asaprimer,producethefirststrandofRNaseH:itisakindofenzymewhichcanpartiallydigestmRNA,yieldingasetofRNAprimersbase-pairedtothefirststrandcDNADNApolymerase:tobuildthesecondstrandcDNAontheRNAprimersTerminaltransferase:addoligo(dC)totheendsofthecDNAcDNAanneal(退火tocomplementaryoligo(dGendsofasuitableUseareverseprimerwithHindIIIsiteatits5’-endtostartfirst-strandcDNAsynthesisPCRreactionusingthesynthesizedcDNAastemplate(forwardprimerhasBamHIsite)CutthePCRproductswithHindIIIandBamHI,ligateitintoavector.RACE:rapidamplificationofcDNAends,amethodforextendingapartialcDNAtoits5’-or3’-end(通過PCR進行cDNA末端快速克隆的技術)TomakethewholelengthofcDNA:5’-RACEand3’-5’-Hybridizethe pletecDNAtomRNA,useRTtoextendthecDNAtothe5’-endofmRNA.AddCresidualstothe3’-endoftheextendedcDNAUseanoligo(dG)primertosynthesizethesecondstrandofcDNA(terminaltransferase)PerformPCRusingknown3’sequenceandoligo(dG)asprimers3’-AsimilarproceduretoextendthecDNAinthe3’-directionUsingananchorprimer(錨定引物)Inthatcase,thereisnoneedtotailthe3’-endofthecDNAwithterminaltransferasebecausethemRNAalreadycontainspoly(A)andtheanchorsequence;thus,thereverseprimerwouldbetheanchorprimer.MethodsofexpressingclonedUsingcDNAsequencetomaketheclones:becausetheintronshasbeenremovedincDNACouldusingantibodytoidentifyclonesfromtheexpressingExpressionvectorshavetwoessentialVectorsproducefusionproteins融合蛋白）Theexpressionproductscontainextraaminoacids.EukaryoticexpressionsystemsAdvantagesof“Eukaryoticproteinsmadeineukaryotic(1)Proteinwillbefoldedproperly;(2)proteinsaremodifiedinaeukaryoticUsingashuttervectorthatcanreplicateinbothbacteriaandeukaryoticcells)theinitialcloningisusuallydoneinE.Coli,thenthe binantDNAistransferredtotheeukaryoticcells.(Yeastexpressionvector,Baculo 桿狀).Tiplasmid:itcantransportforeigngenesintoplantcellsandensuretheirreplicationthereNativeT-DNAplasmid:Crowngall(冠狀腫瘤)CrownGallDiseasehastwomajorelements:(1)tumorgeneswhichsynthesizeextraplantgrowthhormonestostimulateneoplasticgrowthofinfectedcells,and(2)opinegeneswhichsynthesizeunusualaminoacids(opines)thatcanonlybemetabolizedbyAgrobacterium.(Ionexchangechromatography（離子交換層析Gelfiltrationchromatography（凝膠過濾層析DNAandRNAmoleculesarenegativecharged,theymigratetowardtothepositiveTheratesofmoleculesaredifferent.Commonly,smallermoleculesmoveThemobilitiesoffragmentsarecorrelatedwiththelogofmolecularweightorthenumberofGelelectrophoresisusesconstantcurrent(恒定電流PFGE(pulsed-fieldgelelectrophoresis,脈沖場凝膠電泳)wasinventedfortheseparationoflargesizeDNA,thelinearrelationshipbetweenthelogofaDNA’ssizeanditsmobilitynolongerexistswhentheDNAmoleculesareverylarge.LargerDNAmoleculesareveryeasytobreak.Themethodusespulsesofcurrent,withrelativelongpulsesintheforwarddirectionandshorterpulsesintheopposite,orevensideways,direction(PAGE:polyacrylamidegelelectrophoresis,usedtoseparatepolypeptidesaccordingtotheirmass(molecularweight).SDS:treatproteinswithdetergent(SDS)todenature(dissociate)thesubunitsofprotein.SDScoatsallthepolypeptideswithnegativecharges,sotheyallmovetotheanode.Anditmasksthenaturalchargesofthesubunits,sotheyallelectrophoreseaccordingtotheirmolecularmasses,notbytheirnativecharges.Step1:themixtureofproteinsiselectrophoresedthroughanarrowtubegelcontainingmoleculescalledampholytes(雙性電解質),whichsetupapHgradientfromoneendofthetubetotheother.Anegativechargedmoleculewillelectrophoresestowardstheanodeuntilitreachesitsisoelectricpoint(等電點).Step2:Thegelseparatedfromstep1isplacedatthetopofaslabgelforordinarySDS.Proteinswillberesolvedaccordingtotheirsizes.Ion-exchangechromatographyIEC,離子交換層析Usearesin樹脂)toseparatesubstancesaccordingtotheir(SeparatesmoleculesbasedontheirColumnisfilledwithporousresin多孔滲水樹脂whichletsinsmallersubstances,butexcludeslargerones.Andthesmallersubstancesmoveslowerthanthelargerones(Detectionofthetinytiesofsubstancesusedinmoleculebiologyexperimentsgenerallyrequirestheuseoflabeledtracers.RadioactivetracerscanbedetectedbyautoradiographyofliquidscintillationcountingNonradiosctivelabeledtracers:chemiluminescence(化學發(fā)光）NucleicacidhybridizationPrinciple:asingle-strandednucleicacidcanformadoublehelixwithanothersinglestrandofcomplementarybasesequence.Southernblot:identifyingspecificDNAfragmentsGelelectrophoresisofthedigestedDNADNAfragmentsaredenaturedandtransferredintoanitro-UsecertainlabeledprobestohybridizetheWashouttheextraprobe,autoradiographyorothermethodstodetectthelabeledSaliteDNA&DNASaliteDNA:AportionofDNAineukaryoteswhosedensitydiffersfromthatofthemajorityofDNA,andthatconsistsofshort,repeatingsequencesHighlyrepetitive,veryshort,intandemarrays,concentratednearthecentromeresandformsalargepartofheterochromatin,asseparatebandinbuoyantdensitygradient,nofunctionfoundexceptapossibleroleinkinetochorebindingDNAfingerprinting:theuseofhighlyvariableregionsofDNAtoidentifyparticularindividuals,usingaminisa liteDNAasaprobe liteDNA(小DNA):DNAsequenceswhicharehighlypolymorphic,5-100bpupto3000 liteDNA(微DNA):1-5bpsequencerepeatedto50-100bplengthsSSR(Simplesequencerepeat)=STR(ShortTandemRepeat) literepeatsarethebasisoftheDNAfingerprinting(印跡)StepsofDNATheblotwashybridizedwithalabeled DetectionofthelabeledDNADNAty:usingaspecificprobetoidentifythedifferenceamongindividualsinoneANorthernblotissimilartoaSouthernblot,butitcontainselectrophreticallyseparationRNAsinsteadofDNAs.Itisusedtostudytheexpressionofagene,andneedaloadingThehousekeegeneisusedasloadingInsitu-hybridizationISH,原位雜交ISHisusedtolocategenein(HybridizelabeledprobestowholechromosomestolocatespecificDNAInsituRNAhybridization:HybridizelabeledprobestoparticulartissuestoseeexpressionofDNA Usedideoxynucleotides雙脫氧核苷酸）toterminateDNAsynthesis,yieldingaseriesofDNAfragmentwhosesizescanbemeasuredbyelectrophoresis.RestrictionMap:CuttheDNAinquestionwithtwoormorerestrictionenzymesinseparatereactions,measurethesizesoftheresultingfragments.ItisusedtodetermininginwhichorientationtheinsertionwasligatedintotheplasmidMapWhyS1MapLocatethe5’-or3’-endsofRNAs(startingandstoppointsoftranscription)fytheamountofagivenRNAincellsatagiventime.HowdoesitcarriedLabelasingle-strandedDNAprobethatcanhybridizeonlytothetranscriptofinterestHybridizationoftheprobe(single-strandedDNA)tothetranscriptDigestionbyS1nuclease(specificallydegradesingle-strandedpolynucleotides)Thetranscriptshybridizedwithprobewasprotectedfromdigestion(Isolatenucleifromcellsandallowthemtoextendinvitrothetranscriptswhichhadalreadystartedinvivo.Inaisolatednuclei,RNApolymerasethatalreadyinitiatedtranscriptioninvivowill“run-on”,whiletheinitiationofnewRNAchainsdonotoccur.Thisisusedtomeasuretranscriptionrates,orfindwhichgenesaretranscribedinReportergeneAreportgene’sproductisveryeasilytoReporterLacZ(β-Cat(chloramphenicolacetyltransferase氯霉素乙酰轉移酶Otherreporter GFPGreenfluorescentprotein,綠色熒光蛋白)FeaturesofDNAAllDNApolymerasessynthesizeDNAinthe5’to3’direction,readingthetemplate3’toDNApolymerasecannotinitiateDNAsynthesiswithouta10-12ntlongRNAWaysofDNABidirectionalreplication:Tworeplicatingforksmoveinoppositedirectionsawayfromtheorigin.Unidirectionalreplication:Onlyonereplicatingforkmovesfromtheorigin.Rollingcirclereplication:onestrandofadouble-strandedcircularDANremainsintact&servesasthetemplateforelongationoftheotherstrandatanick.ORI:AuniqueDNAsequenceatwhichDNAreplicationisinitiated.Replicon(子):alltheDNAreplicatedfromoneoriginofreplicationProkaryoticchromosomesarecircular.DNAreplicationbeginsatasingleoriginpointandproceedsaroundthecircleinbothdirections.Ineukaryoticcells,thechromosomesarelinear.Theyarealsomuchlarger.Inordertokeepreplicationwithinareasonabletimeframe,theselargechromosomesbeginreplicationatseveraloriginpoints.Eachreplicationoriginproceedsbi-directionally,againwithleadingandlaggingstrandsaccordingtothesamerules.E.Coli’splasmidcolE1containsaEcoRIsite,linerizedplasmidDNAisusedastemplate,itcanbeseenthatthereplicationtakesplaceinunidirectional.CircularDNAcanreplicatebyarollingcirclemechanism:onestrandofadouble-strandedDNAisnickedand3’-endisextended,usingtheintactDNAastemplate.Whenoneroundofreplicationiscomplete,afulllength,single-strandedcircleofDNAisreleased,whichiscalledasσmodel.EnzymologyofDNADNADNAHelicase（DNA解旋酶DNAhelicase:theenzymethatharnessesthechemicalenergyofATPtoseparatetwoparentalDNAstrandsatthereplicatingfork.Thereareabout10proteinsinE.colithathavehelicaseactivity.OneinvolvedinDNAreplicationistheproductoftheDnaBgene,whichisessentialforDNAreplication. SSBPisakindofspecificproteinwhichcankeepthesinglestrandedDNAfromcoilingupandinhibittheprogressofthereplicationenzymes.SSBstimulatesDNAsynthesisandprotectthesinglestrandedDNAfrom(DNAligaseisenzymecatalyzingthereactionduringwhichfragmentsofDNAarejoinedtogether.ThistakesplacewhentheOkazakifragmentsarereadytobelinkedtogether.FirsttheRNAprimerisremovedandDNAsynthesizedinitsplace.(Itisusedtounwindahelixforreplicationtoproducetopologicalstrainandsupercoiling.Thisstrainmustberelieved,orthereplicationcannotcontinue.TypeIThesearealsocallednicking-closingenzymesandfunctiontorelaxsupercoiledcircularmolecules.TheseenzymesfunctionwithouttheinputofenergyintheformofATP.Theenergyisprovidedbythereleaseofstraininthemolecule.TypeIItopoisomerasesarealsocalledDNAgyrases(促旋酶).TheseenzymescaneitherrelievesupercoilingorintroducesupercoilingintoaDNATheyrequiretheenergyofATPfortheiractionDNAgyrase:atopoisomerasethatpumpsnegativesuperhelicalturnsintoDNAtorelaxthepositivesuperhelicalstraincreatedbyunwindingDNAduringDNApolymeraseI:RemovingofRNAab

5’ParentalDNApolymeraseSimultaneousremovalofprimerandsynthesisofDNAtotillinthegapcd

DNA

RNAsynthesisisnotprimer-dependent,theprimerissynthesizedunderthecatalysisofRNAprimaseorprimase.Primaseiscontainedinastructurecalledtheprimosome(引物合成體whichiscomposedof6proteins,n,n’,n’’,DnaB,CandI.Whenanewlysynthesizedlaggingstrandhasbeencopiednearthe3'endofonestrand,youneedtohaveaprimertostartanotherlaggingstrand.Buthowdoyoudothisbeyondthe3'endofthechromosomestrand?omerasesisjusttheenzymewhichisrelatedtoreversetranscriptase,inwhichthereisasmallRNAtemplateasapartoftheenzymestructure.omereshaverepeatedsequencescalledomererepeats.TheRNAtemplateofomeraseisusedtoaddaomererepeattothe3'end.AnRNAprimeristhensynthesizedasacopyofthisextensiontotheDNAandthelastlaggingstrandismade.LatertheextensionistakenalongwiththeRNASummaryofatypicalprokaryoticreplicationReplicationforkmovesdownalongtheDNAasthehelixisunwoundunderthecatalysisofhelicaseandtypeItopoisomerase.Re-annealingoftheseparatedDNAstrandsispreventedbytheactingofSSBP.BothleadingandlaggingstrandsaresynthesizedbythedimerofDNApolIII.Continuousreplicationisgoingonforleadingstrandusingthe3’-5’singlestrandastemplate.ThelaggingstrandisloopedaroundtheDNApolymeraseIII,whichallowsonelargepolIIIcomplexconsistingofapairofsubunitstopolymerizebothleadingandlaggingstrandssimultaneously,thenreplicationofbothstrandsistherebypreciselycoordinated.PrimosomemovesdownthelaggingstrandanddiscontinuouslyproducesRNAprimer.OkazakifragmentisproducedusingthisRNAprimerbyDNApolIII.WhentheDNAfragmentbuttsupthenextOkazakifragment,RNAprimerisdigestedbyRNaseHandthenickissealedbyDNAPolI.ThegapbetweenOkazakifragmentsissealedbyDNAligase.GenestructureinOperon(元):anunitofgeneexpressioninprokarytoeswhichincludesstructuralgenesandcontrolelementsCistron順反子AsectionofDNAthatcontainsthegeneticcodeforasinglepolypeptideandfunctionsasahereditaryunit.Promoter(啟動子ThatisaDNAsequencetowhichRNApolymerasebindspriortoinitiationoftranscription.Itisusuallylocatedjustupstreamoftranscriptionstartsite.-35box:TTGACA,centeredin-35bpUPelement:somepromoterhasanextraelementin-40~-Theclosertotheconsensussequence,thestrongerthatpromoterwillThetranscriptioninitiationsiteisapurinein90%ofgenes.MoreG>ATranscriptioninRNARNApolymerasecontains:2largesubunits(β,β’)andσ&αsubunits.RNApolymeraseholoenzymeincludesβ’,β,σ,α2CoreenzymeiswithoutCoreenzymecontainsthebasictranscriptionmachinery,anditonlytranscribenickedDNAtemplate,notintactDNA.Withoutσfactor,thecoreenzymewilllosespecificity.BindingofRNApolymerasetoHoloenzymesearchforabindingsiteontheBoundBoundingtightly,meltingDNAandforminganopenpromotercomplexσSummaryforσHelpRNApolymerasebindstoapromoterThetightbindsneedtomelttheDNA,soσfactorcanselectgenetotranscribeAftertheinitiation,σfactorisboundlooselyintheelongatingElongationoftranscriptionneedscoreenzyme,andDNAunwindingneedsTheroleoftheαsubunitinUPelementrecognition:withoutafunctionalαsubunit,UPelementisnolongeranenhancer.βinphosphodiesterbondformation:βsubunitdeterminingtherif()RifblockthetranscriptionStreptodigin(利鏈菌素)blocksRNAchainelonglation,andthecommonthingforinitiationandelongationisphosphodiesterbondformation.βsubunitensuresformationofphosphodiesterbondTwoRNApolymerasebindingsitesexistonDNA,βandβ’arethesubunitsbindingρ(rho)independentAninvertedrepeatallowsahairpintoformattheendofthetranscription,andastringofTsinthenon-templatestrandresultsinastringofweakrU-dAbasepairsholdingthetranscriptiontothetemplatestrand.Modelofρ-independentThepolymerasehaspausedatastringofweakrU-dAbasepairs,andahairpinhasstartedtoformjustupstreamofthesebasepairs.Asthehairpinforms,itfurtherdestabilizestheRNA–DNAhybrid.TheRNAproductandpolymerasedissociatecompleyfromtheDNAtemplate,terminatingtranscriptionρasaterminationTheMechanismof

Rhoconsistsofahexamerofidenticalsubunits,eachofwhichhasATPaseactivity.Rhoisnotapartofthepolymerase.Itbindstothetranscriptupstreamoftheterminationsiteatarholoadingsite.BindingofrhotoRNAactivatestheATPase,whichstheenergytopropeltherhohexameralongtheRNA,followingRNApolymerase.ItcontinuesuntilthepolymerasestallsintheterminatorregionjustaftermakingtheRNAhairpin.Thenrhocancatchupandreleasethetranscript.RhohasRNA–DNAhelicaseactivitythatcanunwindanRNA–DNAhybridRegulationoftranscriptioninControltheexpressionofgenes:someon,someOperon(元):Thisisaunitofgeneexpressioninprokaryotes,whichincludesstructuralgenesandcontrolelements.Eachoperonissubjectedtostringentregulation,on(increasedtranscriptioninitiation)oroff(decreasedtranscriptioninitiation).Theregulationisdependentontheneedsofmicroorganism.Lacoperon乳糖子Araoperon糖子Trpoperon色氨酸子Polycistronicmessage(多順反子):anmRNAbearinginformationfrommorethanonegeneOperator(子):ADNAelementfoundinprokaryotesthatbindstightlytoaspecificrepressorandtherebyregulatestheexpressionofadjoiningControlsoflacNegativecontrol:thebindingofrepressortotheoperatorkeepstheoperonPositivecontrolcatabolite(代謝副產物activatorproteinCAP)inconjunctionwithcyclic-AMPstimulatestranscription.StructureoflacLacI:GeneencodingtheLacrepressor(LacR)P:Lacpromoter(bindingsiteforRNApol)O:Lacoperator(bindingsiteforLacR)lacY:totransportlactoseintolacZ:breaklactoseintogalactoseandlacA:unknownfunction,involvinginmetabolismofTwokeyelements:repressorandoperator.Theoperonisturnedoffaslongastherepressorbindstotheoperator.Whenglucoseisexhaustedandlactoseisavailable,thefewmoleculesoflacoperonenzymesproduceafewallolactosefromlactose.Andtheallolactoseactsasaninducerbybindingtherepressor,andtherepressordissociatesfromtheoperator.Theoperonnowisturnedon.Mutationinrepressor(I-),whichproducesnorepressor,isMutantrepressorgene(I-d),ofwhichrepressorscannotbindtooperator,isdominantnegative.Mutationinrepressorgene(Is),whoseproductcannotbindtoinducer,iscis-andtrans-dominant.Mutationinoperator(Oc),ofwhichtheoperatorcannotbindtorepressors,iscis-dominant.PositivecontrolofLaccAMPisresponsetoglucoseCAP(cataboliteactivatorprotein,分解物激活蛋白)conjugatedwithcAMP(環(huán)腺苷酸）stimulatestranscription.AndcAMPconcentrationdependsonglucose,moreglucoselesscAMP.TheLacoperonisactivatedonlywhenglucoseconcentrationislow.SummaryofLacLacstructuregenesonlyexpressedwhen:NoGlucoseandlactoseAraPositivecontrolofaraoperoniseffected plex,whichissimilartoLacAraCproteinisusedasrepressor,whichbindstooperatorsintwositesandloopstheDNA.ArabinoseactsasTheyaregenesforenzymesmakingtryptophan.Therepressionrequiresthebindingoftrpandapo-repressor(輔阻遏蛋白).Apo-repressorisinactive,unlessitbindstotryptophan,whichactsasarepressor.Lowtrp,norepressionHightrp,AttenuatorisaregionofDNAintheupstreamofoneormorestructuregenes,whereprematuretranscriptionterminationcanoccur.Whentheconcentrationoftrpislow,thereexistlesstRNAtrpincells,andtheremovalofribosomethroughtrpcodonsisveryslow.SothemRNAcouldnotformhairpinstructure,andthe