分子生物学全

KillingMessengersDoublestrandedRNAmediatesthesilencingofthetranslationofitscorrespondingmRNA杨官品中国海洋大学海洋生命学院电话-mail:yguanpin@P5’OH3’P5’OH3’Shortinterfering(si)RNAMolecularhallmarksinclude5’phosphorylatedends,a19ntduplexregion,and2ntunpairedandunphosphorylated3’andsThisstructureischaracteristicofanRNaseIIIlikecleavagepattern,whichledtotheidentificationofthehighlyconservedDicerfamilyofRNaseIIIenzymesasthemediatorsofdsRNAcleavage.ATPADP+ppiDicer21-23ntdsRNAsiRNADuplexThisprocessisrestrictedtothecytoplasm.Asthefirststep,digestlongdsRNAtoproducesiRNAsDicerfunctionsinanATP-dependentmanner.dsRNAMultiproteinRNAinducingSilencingComplex,RISCSelfassemblingATPADP+PPiRISCactivationSensestrand

siRNAisincorporatedintotheRNAinducingsilencingcomplex(RISC).Thisstepisenergyfree,theuptakeofsiRNAbyRISCisindependentofATP.TheunwindingsiRNAduplexrequiresATP.Onceunwound,thesinglestrandedantisensestandisholdbyactivatedRISCandthesensestrandisreleased.TheantisensestrandwillserveastheguidertoleadRISCtothetargetmessengerRNAwhichhasacomplementarysequence,resultingintheendonucleolyticcleavageofthetargetmRNA.mRNAm7G(A)nThereisastrictrequirementforthesiRNAtobe5’phosphorylatedtoenterintoRISC,andsiRNAsthatlack5’phosphatearerapidlyphosphorylatedbyanendogenouskinase.ThedssiRNAisunwound,leavingtheantisensestrandtoguideRISCtoitshomologoustargetmRNAforendonucleolyticcleavage.ThetargetmRNAiscleavedatasinglesiteinthecenteroftheduplexregionbetweentheguidesiRNAandthetargetmRNA,10ntfromthe5’endofthesiRNA.ArethereendogenoussiRNAs?

Interestingly,endogenouslyexpressedsiRNAshavenotbeenfoundinmammals.However,therelatedmicro(mi)RNAshavebeenclonedfromvariousorganismsandcelltypes.miRNAsareshortRNAspecies,about23ntinlongth,whichareproducedbyDicercleavageoflonger,about70nt,endogenousprecursorswithimperfecthairpinRNAstructures.

ThemiRNAsarebelievedtobindtothesitesthathavepartialsequencescomplementaryinthe3’untranslatedregion(UTR)oftheirtargetmRNA,causingtherepressionoftranslationandtheinhibitionofproteinsynthesis.

InadditiontoDicer,otherPAZ/PIWIdomainprotein(PPD)includingeukaryotictranslationinitiationfactor2C2(elf2C2)arelikelytofunctioninbothpathways.HairpinmiRNAprecursorItmaybeanubiquitous,non-specificregulatingfactorattranslationstep.Wearehappytofindthatcellshaveevolvedandkeptsuchaway,otherwise,oursiRNAcouldnotcomeintobeing.HairpinprecursorDicerSinglestrandedmiRNAAproteincomplex,PPDmiRNAproteincomplex(miRNP)miRNAmediatedtargetrecognitionmRNAm7GpolyAUntranslatedregionTranslationinhibition

miRNApathway,existsnaturally.AlthoughoriginalyidentifiedonthebasisofitsabilitytoprocesslongdsRNA,Dicercanalsocleavethe~70ntmiRNAprecursortoproduce~22ntmiRNA.UnlikesiRNA,miRNAissinglestranded.ItincorporateintoamiRNAproteincomplex(miRNP),whichpairswithpartialsequencecomplementarytotargetmRNA,leadingtotranslationrepressionwithoutdigestionofmRNA.InadditiontoDicer,thetwopathwaysrequireotherPAZ/PIWIdomainprotein(PPD)includingeukaryotictranslationelogationfactor2C2(elF2C2).RNAimediatedbytheintroductionoflongdsRNAhasbeenusedasamethodtoinvestigategenefunctioninvariousorganismsincludingplants,hydras,trypanosomes,drosophila,mosquitoes,andmouse.andFIsh?andChina?TheapplicabilityofthistoolinmammalsislimitedbecausetheintroductionofdsRNAlongerthan30ntinducesasequencenonspecificINTERFERONresponse.InterferontriggersthedegradationofmRNAbyinducing3’-5’oligoadenylatesynthase,whichinturnactivateRNaseL.(toallmRNAspecies0Inaddition,interferonactivatetheproteinkinase,PKR,whichphosphorylatesthetranslationinitiationfactorelF2a,leadingtoaglobalinhibitionoftranslation.dsRNAsdonotworkinmammals,howaboutsiRNAdirectly?WhenchemicallysynthesizedsiRNAswereintroducedco-transfectionallyintofruitflycellswithluciferasereporterconstruct,theyarefunctional.TheresultsarecomparablewiththoseobtainedwithlongdsRNAs,causingsequencespecificlossoffunction,thesilencingofluciferasegeneexpression.siRNAtransfectioncansilencetheendogenousnuclearenvelopeprotein,LaminA/C,inmammaliancellswithoutactivatingnonspecificeffects.Thesefindingshaveledtothewidespreaduseofthistechnologytostudythegenefunctionincludingtargeteddisruptionofclinicallyrelevantgenes,inludingthepotentialtherapeuticapplicationofRNAibasedtechnologies.LongdsRNAinsomeorganismsotherthanmammals,andsiRNAtransfectioninmammals.

siRNAMethodstogenerateshortRNAsthatsilencegeneexpression

--SilencingbyRNAsthataregeneratedinvitroLongdsRNAsiRNAbasedhairpinmiRNAbasedhairpinsiRNAsiRNAmiRNASinglestranded

Chemicallysynthesized,bypassdicing,incorporatewithRISC,targetmRNAcausingdegradationdsRNA,introducedintocells,dicedintosiRNA,silenceRNAtranslationPerfectduplexhairpinRNA,dicedintosiRNA,silencemRNAImperfectduplexhairpinRNA,basedonpre-microRNA(miRNA)structure,dicedintossmiRNA,directgenesilencingbybindingtountranslatedregionofmRNAs

SilencingbyshortRNAsthataregeneratedinvivo:a)LonghairpinRNAexpressedfromRNAPolIIpromotor,yieldingapopulationofsiRNAwithserveralsequencespecificities.polyAboxPolIIAAAAAAADicerDssiRNAsb)TandempolIIIpromotersthatexpressindividualsenseandantisensestrandsofthesiRNAsthatassociateintransTTTTTTTTPolIIIPolIIIuuuuuuuuuuuuuuuusiRNAc)AsinglepolIIIpromotorthatexpressesshorthairpin(sh)RNAwiththesenseandantisensestrandsofthesiRNAthatassociateincis

PolIIIDicersiRNAPerfecthairpind)Imperfectduplexhairpinstructure,pre-miRNAstructure,canbeexpressedfromaPolIIpromotoranddicedintomaturemiRNAwhichcandirectgenesilencing

PolIIpolyAboxm7GpolyAmiRNAAirBreakOperonsandthecontroloftheirexpression

（表达调控单元及其表达调控）一个Operon本来是可以为RNA聚合酶转录表达的，但因为Repressor的存在而不能转录了，这样的调控形式称负控制，从有到无的控制。Fromworkabletounworkable,negativecontrol.仍然是以operon的表达为落脚点，在其他都正常的情况下，某小分子的存在使负控制的Operon表达，称可诱导的负控制(Induciblenegativecontrol)。诱导物不是乳糖Lactose，而是半乳糖苷Galactose。因为控制有泄漏Leakage，因此，半乳糖苷酶有衡量Traceamount表达，只要有乳糖存在，就能合成足以解除负控制的半乳糖苷。I基因编码Repressor，组成型表达，基因可以与O/P临近，也可以空间上分离；控制者元件Operator和促进者Promotor元件可以相临，部分重叠或者完全重叠或空间上分割。*Thehypothesis(acompetitionbetweenRNApolymeraseandrepressor)mayexplainthecontroloflacoperon.*However,thecontrolmechanismdoesnotstophere.*Howtoresponsetoglucose?AheadoftalkingaboutthisHOW,AuxiliaryoperatorO3

PromotorMajoryoperatorO1

AuxiliaryoperatorO2

LacZ1.0000.3380.5380.0140.0010.0010.0010.001RepressionbychromosomalI+TetramerofrepressorTetramerofrepressorTetramerofrepressorO3O1O2PromotorLacZ**Cyclic-AMP**Cataboliteactivatorprotein;CAP/Cyclic-AMPreceptorprotein,CRP/officialnameofthegene,crp

**cAMPisnegativelyproportionaltoglucose**Incaseoftheexistenceoflactose,lacoperonispositivelyregulatedbythebindingCAP**cAMPcaninitiatesuchpositiveregulation,therefore,itisaninducer,andsuchregulationisinduciblepositivecontrol**GlucosecannottouchCAPdirectly,therefore,wenevertalkifitisaneffector(inducercanrefertogalactose,butrepressoronlytotheprotein).“inducible”or“repressible”wasdefinedaccordingtothedirectsmallmolecules,effectororinducer.**Towhattypeofcontrollingmechanismthelacoperonbelongs?Distinguishingpositiveandnegativecontrol+/-controlsystemsaredefinedbytheresponseoftheoperonwhennoregulatorproteinpresents.Genesundernegativecontrolareexpressedunlesstheyareswitchedoffbyarepressorprotein(表达变不表达).Negativecontrolprovidesafail-safemechanism:iftheregulatorproteinisinactivated,thesystemfunctionsandsothecellisnotdeprivedoftheseenzymes.Forgenesunderpositivecontrol,expressionispossibleonlywhenanactiveregulatorproteinpresents(不表达变表达).Themechanismforcontrollinganindividualoperonisanexactcounterpartofnegativecontrol,butinsteadofinterferingwithinitiation,theregulatorproteinisessentialfortheoperon.ItinteractswithDNAandwithRNApolymerasetoassisttheinitiationevent.Repressor/activator.Otherpositivecontrolsprovideforglobalsubstitutionofsigmafactorsthatchangetheselectionofpromotors,orantiterminationfactorsthatchangetherecognizationofterminators.Inducibleorrepressibleoperons;operonisthetarget.Definedbythenatureoftheirresponsetothesmallmoleculethatregulatortheirexpression(direct).Inducibleoperonsfunctiononlyinthepresenceofthesmallmolecularinducer;Repressibleoperonsfunctiononlyintheabsenceofthesmallmolecularcorepressor(socalledtodistinguishitfromtherepressorprotein).Theterminologyusedforrepressiblesystemsdescribestheactivestateoftheoperonsasderepressed;thishasthesamemeaningasinduced.Theconditioninwhicha(mutant)operoncannotbedepressedissometimecalledsuper-repressed;thisistheexactcounterpartofuninducible.Inductionisachievedwhenaninducerinactivatesarepressorproteinoractivatesanactivatorprotein;Repressionisaccomplishedwhenacorepressoractivatesarepressororinactivatesanactivatorprotein.****Anoperonmayhavetwotypesofcontrolatthesametime.Forexamplelacoperon.****WhatleftforyouistoreadtheBookinEnglishinordertounderstandtheconclusiontocompletion.

**被调控单元Regulon,forexample,ThemalRegulon;malmeansmaltose;**CAPmediates,therefore,thepositiveregulationmechanisminvolved;However,thisistoresponsetoglucose;Withoutglucose,CAP-cAMPisthere,whenmaltoseexists,theregulonexpresses;Otherwise,not.Quistionhereis“how”?Thesameaslacoperon?**Maltosemetabolizingneedssetsofenzyme;theseenzymesarenotcontiguous;Instead,theyaregroupedandcontroledbymultiplepromotors;**Regulon,asetofnoncontiguousandcoordinatelycontroledgenes.Controledtogather,butseparatedspatially.**Maltoseisanalternativeenergyofglucose;theregulonmustresponsetoglucosefirst;Therefore,CAPmustinvolveinsuchregulation.**Inanovelway;**WorksinassociationwithmalT,alsoregulatesthemalpromotors;**malTrequiresATPandmaltotriose,maltosereguloninducer;malpromotorsDependonmalEp

malKp

pulAp

pulCp

pulPp

pulTp

CAP+MalTCAP+MalTMalTMalTMalTCAPOnepromotoriscontroledbyCAPisenoughfortheresponsetoglucose+----/+-/+malKpmalEpEFGKBM1013CAP-cAMPMalTMalTaraOperon**CAPsharesitsbindingsitewithRNAPataraPcpromotor;RNAPcompeteswithCAP-cAMP;RNAPhavethechancetotranscribearaC.**Theresponseisstrong;araCshouldbetranscribedorclosedeffectively;itsexpressionshouldbecontrolled.**Serveastherepressorofitsownexpression;Autoregulation.(aproteincontrolsitsownsynthesis,moreexampleslater)**araoperonisalsoacataboliterepressibleoperon;**twooperators,O1andO2,controlsthetranscriptionofcontrolgene,araC,andPBAD,265bpupstream;**CAPbindingsiteis200bpupstreamofthePBAD,stillworks;**Theoperonhasanothersystemofnegativeregulation,mediatedbytheAraCprotein.araPcaraPBADaraO1araO2araCaraII2I1AraC/arabinose-AraCAutoregulationofaraC

AraCbindstoaraO1andpreventstranscriptionleftwordfromPcthroughthearaCgene.ThiscanpresumablyhappenwhetherornotarabinoseisboundtoAraC,thatis,withthecontrolregioneotherunloopedorlooped.trp(prononced“trip”)operontrpoperoncontainsthegenesfortheenzymesthatthebacteriumneedstomaketheamonoacidtryptophan.Controlofthetrpoperonbyattenuationtrpoperonemploysanothermechanismofcontrolcalledattenuation.Why?Responssionofthetrpoperonisweak-muchweakerthanthatoflacoperon.Considerablereanscriptionofthetrpoperoncanoccureveninthepresenceofrepressor.Infact,intheattenuatormutantswhereonlyrepressioncanoperate,thefullyrepressedleveloftranscriptionisonly70-foldlowerthanthefullyexpressedlevel.Theattenuatorsystempermitsanother10-foldcontrolovertheoperon’sactivity.Intotal,700fold.Thisisvaluablebecausesynthesisoftrptophanrequiresconsiderableenergy.trpL(leader)attenuatortrpO/PtrpEmRNAAUGstartUGAstopMetSer14Leader,peptideWhy/Who?AUGAAAGCAAUUUUCGUACUGAAAGGUUGGUGGCGCACUUCCUGAACCACUUAUGUGACGGAGCCCGCCUCGGGCAGGUUUUUUUAUGAAAGCAAUUUUCGUACUGAAAGGUUGGUGGCGCACUUCCUGAUAUGUGACGGGCAUACCCAGCCCGCCUAAUGAGCGGGCUUUUUUUURNAPribosmePhageStrategiesMolecularBiologyofPhagesUnderstandingthelifeofphageatmolecularlevel!DNALyticCycleLysogenyPhageDNAisintergratedintobacterialgenome;bacterialivehappilyeverafterLysogenicbacteriaareimmunetoFurtherinfectionInductionPhageDNAisreleasedandenterslyticcycleLyticdevelopmentinvolvesthereproductionofphageparticlesandthedestructionofthehostbacterium,butlysogenicexistenceallowsthephagegenometobecarriedaspartofthebacterialgeneticinformationWhyandhow?Descriptionatmolecularlevel.基因组最小接管一切细胞资源，如何做到？谁聪明，谁进化上更高级？12ntterminalrepeatComplementaryeachotherCossiteCohensiveendsHostLigaseHostbacterialchromosomeCohensiveendsGenome;genomesize;andgenomecomplexityInchromosomes,22+X+YofhumangenomesReplication:rollingcyclestyle

HostbacterialDNAreplicationsystemcoscosAgenePackase●●PackageisaselfassemblingprocessOtherstructuralparts5‘biooperongaloperonattachmentsite

attBattPBOB’POP’PhageattLattRBOOP’PB’BOB’+POP’

BOP’+POB’int+IHFint+xis+IHF+?FIS)int,intgrase;IHF,integrationhostfactor;FIS,factorofinversionstimulation;xis,excisionaseMolecularmechanism?整合效率远远低于复制可逆的，在没有阻抑蛋白时，一个的整和不等于其它Homologousrecombination;geneknocknout;insertionalmutagenesis;transposon;puc19(lacZ);heterologouscharomatinactivation;etc.GCTTTTTTATACTAACGAAAAAATATGATTGCTTTTTTATACTAACGAAAAAATATGATT235bp23bpBB’4bpOO110bpPP’GCTTTTTTATACTAACGAAAAAATATGATTGCTTTTTTATACTAACGAAAAAATATGATTO,coresequence,15bpinlength,ATrich;Breakageandlinkageincoresequence,likerestrictioncleavage,arrows,thesitesofbreakageattBandattPareindifferentlengths,235and23bprespectively,indicatingthattheyplaydifferentrolesinrecombination;WhenusingsupercoiledDNAassubstrates,thesupercoilconformationwaspreservedinproducts,indicatingthatnofreeendsappeared,topo-isomerasemightinvolvedinthisprocess,atopologicalshift,notbreakingandlinkingprocesses.RecombinationsubstrateandintandIHFdosagesareproportionalifbeingisolated,indicatingthatintandIHFarenotcatalysts,rathertheyareconstructstopoisomeraseCuttingandligationinonestep

Transcriptionfirstorreplicationfirst?Immediatecycling;Ifreplicationfirst,fromwhere,thespecificproteinscome;Youmayask,thereplicationproductsmightbecutandlinkedintothecycle,however,pleasebearinyourmindthatthisstepneedpackaseandthepackaseshouldbefixedinthepre-structure,ortheprecursorofthephageparticle,wherearethey;Theotherreasonthatthetranscriptionshouldbefirstisthatthereplicationneedcloseandusehostsystem,whotellshosttodothis,thetranscriptionproductsfromthephage,someproteins;Thisisjustmyunderstanding.Bacteriawithintegratedphageisimmunetotheinfectionofthesamephagespecies.Why?

Evenastheprovirusthatintegratedintothebacterialchromosome,thecIproteinissynthesizedconstitutively;newlyenteredphageDNAwillbeblockedatPRandPLimmediately,andnoproteinsinvolvedinrecombinationorlysiswillbesynthesized.ThenewlyinfectedphageDNAwillbekeptnative,originalandorjustcycled,whichwilldisappearwiththeproliferation,theseparationofbacterialcell,aprocesscalledbiologicaldilution.How?Understandable?Whatisourunderstanding?Themostperfectexampleofunderstandinglifeatmolecularlevel!Ifyouunderstandthelifeofphageatmolecularlevelto80%,thenyouunderstandthemolecularbiologyto99%!Thisismyideaonly.cIcINNcIIIcIIIcrocrocIIcIIOPQReplicationSRALysiscossiteaBrxisintattRecombinationWBLNu3OEFIFIIZUVGTHMLKIJHead&TailProteinsPRMPLOLTLnutL★★ImmediateearlyDelayedearlyPRORTR1nutR★◆◆◆PREHostTR3PR’LateStageTermination?◆HomologousrecombinationDelayedearlyDegreeandamountcis-elementsRegulatorgenesNewRNApol?Highefficientpromoter?RegulationOperon?Moregeneinformationisonthecontinuouslytranscribedstrand.Why?Itismyunderstanding!5‘3‘5‘3‘基因、基因元件、基因表达调控元件在DNA上，包括两条DNA单链；基因表达调控元件与基因在同一DNA分子上，称cis-elements;基因工程（半、准）中，一些基因或元件（如cI,P/O,T等）已被运用；Template,antisensestrand/coding,sensestrand;Heavy/lightstrand;基因写法，coding/sensestrand,与mRNA共线性，基因在DNA上；DNA复制时，若另一链是连续合成的，遗传信息多（做模板机会多，少突变机会），对滚环复制而言，不具有统计意义；溶原/裂解途径的建立依赖PcII-PcIII调节蛋白组作为或者不作为；PcII-PcIII调节蛋白组作为或者不作为又依赖宿主细菌是否让其作为；宿主细菌是否让其作为依赖宿主细菌的基因型和生理状态；当无作为时，…，Pcro关闭所有极端早期、延缓早期和cI基因转录，进入复制后，后期转录停止，复制裂解相关蛋白够了，溶原相关蛋白也够了，因为整合涉及底物、切与连等多步骤、低浓度，时间长或者机会少，因此，复制、裂解优先；机会少不等于不发生，因此，具体到一个噬菌体，谁也说不准具体去向，这里的结论只是统计意义上的结论。当有作为时，…，因为在转录物的前面，应该有多的机会获得足够的量先发生作用。发生作用时，先建立阻抑物蛋白的表达，然后自主调控，不断表达，在复制、裂解相关蛋白足够之前、后期转录开始或者未开始之前，有能力关闭所有的转录，即使后期有，也很快因PQ减少而停止；在生死攸关的时候，整合体系在不足够的情况下，可以通过时间的延长和复制系统不作用时容许这样的等待存在的情况下，总有机会整合进宿主染色体，进入溶原状态，一个具体的噬菌体也可能由此消失（生物稀释作用）。这里说的仍然是统计意义上的结论。有作为时，就开始用反义RNA的形式蹜蹜减少PcII和Pcro；因为只要启动了PRE,进一步的转录就不必要了，解除Pcro可以解除对PRM的限制，Pcro对左右转录的限制，可以有PcI取代；唯一DNA的两条链都被使用的例子，人为的使用例子很多；cI自主调控能成立时，PRE早就不需要了；RNAPol双向等机会；对PcI能发挥作用的噬菌体，同种或含有相同调节蛋白的，具有免役作用；只要解除PcI的作用，就能重新启动转录，面临重新决定，因为PcI的作用都能解除，PcII/PcIII不作为的机会多，但具体到一个噬菌体，谁也说不准；机会少，但一旦有作为，就重新维持溶原状态；一旦无作为，复制裂解、重组整合的相关蛋白都足够，等在那里耗损时间，一旦切下来，立即进入复制裂解周期。**Canweunderstandthisregulationprocessdeeperorindetail?Yes,butlimited.**Isitthebestexampletounderstandthelifesecretatmolecularlevel?Yes.**Whenandhowcanweexplainalllifeprocessinsuchawayaswedoforthephage?Godknows!**WehaveknowntheDNAsequenceofhumangenome,doyouknowhowfarareweinunderstandingGods’designing?101000kms!**YoucansynthesizeDNAorrecoverDNApossibly,canweregeneratelife?Whenwewillhavesuchability?No.1010000yearslater.

cIprotein,therepressor,isadimerDimerizationDNA-bindingConnectorNC92aa11aa236aa132aaDNANproteinAporepressor=repressorDimerMonomerDimer+DNADimerDimer+DNACleavageLysogenyInductionWhyandhowdosetheconcentrationofPcIisreduced?CommunicationbetweenPcI,phageanditssurroundingenvironmentDimerismaintainedinafixedconcentration,~4x10-7M,otherwise,inductionmaystart.调节蛋白/负控制/通过降解实现变构/导致原因不存在？表达？UV？pcI,therepressor,bindsateachoperatorcooperativelyusingahelx-turn-helixmotif12345CterminaldomainstructureisunknownNterminaldomainconsistsof5a-helicesHelix3istherecognitionhelixTACCTCTGATGGAGACCTATCCCTTATAGGGAACGlnSerGlyValGlyAlaLeuPheAsnRepressor-OR1GlnSerAlaIieAsnLysAlaIilHisLysAspAlaValSerGluGlnLysThrAlaLeuGluThrGlnCRO–OR3OR1OR3Pcrowillbedescribedlate;Wecannotshowthedetailsandconformationalshiftinghere;Imageitamouthandmeat,wecannotseethemoviepresentingyouhowalionkillsazebraandenjoysitsmeal.TTTCTTTTTTGTGCTCATACGTTAAATCTATCACCGCAAGGGATAAATATTCTAACACCGTGCGTGTTGACTATTTTACCTCTGGCGGTGATAATGGTTGCATGAAAGAAAAAACACGAGTATGCAATTTAGATAGTGGCGTTCCCTATTTATAAGATTGTGGCACGCACAACTGATAAAATGGAGACCGCCACTATTACCAACGCATOR3OR2OR1RNApolymerasebindingsitePRRNApolymerasebindingsitePRMpppAUGcromRNARepressorproteinLysLysLysThrSerMetNH2AAAGAAAAAACACGAGUApppcImRNApcrorepressorTheaffinityofpcItoOR1is~100foldshigherthanthattoOR2;UponbindingofpcItoOR1,theotherpcIwillbindtoOR2concertedly;Usually,OR3isnotoccupiedbyanypcI;However,whenOR1mutated,OR3willbeused,atthismoment,pcIsynthesiswillbeblocked,andthatisthesignalofenteringlysispathway;Differentaffinitieswereofferedbythesequencedifference;TheaffinityofpcrotoOR3is~1000foldshigherthanthattoOR1andOR2;Needmorepcrothanrepressortocloseleftandrighttranscription;Inthiscase,enoughpQproteinwillbethere,sothatlatephasecouldbeinitiated,integrationwillbegivenup.PR/ORcro

PL/OLcI

PREcIIIcIIRNApolymerasecIdimerPRMpcIIisveryunstable,itiseasytobecleavedbyhostHflA;theroleofpCIIIistoprotectpCIIagainstsuchdegradation.ThetranscriptionbyPREiscrosscro,thewrongdirection,theonlyexampleIsee,butonlyonestrandisusedastranslationtemplate.Moreimportantly,itistheantisenceRNAofcro,whichwillinhibitcrotranslation.PREis7-8foldmoreefficientthanPRM,becausePREhasRBS,andPRM

startsatAUG.

PREhasweakconsensusat–10andlackstheconsensusat–35.RNApolcannotbindtoitalone,however,whenpcIIpresents,itcandoso.+10–10–20-30-40-50startpointBoundbyCIIandRNApolATCTAAGGAAATACTTACATATGGTTCGTGCAAACAAACGCAACGTAGATTCCTTTATGAATGTATACCAAGCACGTTTGTTTGCGTTGCTAATATACAGTTUsualsequencesat–10and-35RNApolcannotbindtoPREbecausethebindingsiteisnottypicalWhenbehelpedbyCII,itcanbindtoiteffectivelyPositivecontrol可阻断正控制Pcroisarepressorneededforlyticinfection**ItinhibitstheexpressionofearlygenesfrombothPLandPR;**TheaffinityofpcrotoPR3isgreaterthanthattoOR2orOR1.ItinhibitsRNApolfrombindingtoPRM(itsfirstaction);**pcrobindstoOR2orOR1.Itsaffinityforthesetwositesissimilar.Thereisnocooperativeeffect.**ItspresenceateithersiteissufficienttopreventRNApolfrombindingfrombothpromoters;Thisinturnstopstheproductionoftheearlyfunction,includingpcroitself(itssecondaction);**BecauseCIIisunstable(如果稳定就是另一种情况),anyuseofPREisbroughttoahalt.Thus,thetwoactionsofpcrotogetherblockallproductionofrepressor.**ItsaffinitytoOR3iseighttimeslowerthanthatofrepressor;pcrowillnotworkuntilenoughpQispresent.Atthattime,lategenesstarttoexpress.Recombinationismoredifficult(oneDNAcopy),replicationstarts.Conceptswecanseehere:**HostligasemakesthephageDNAacycle;**Determinationoftwopathwaysisnotcompletelyclear,Howphageandhostinteract?Nodetailsareavailablecurrently;**Synthesisofproteinfirst,thenreplication,smartdesigningofgeneticinformation;**Latetranscriptionneedaterminator?Smart.**Genomesizeandgenomecomplexity;**Quantitativeandaffinityviewinunderstandingthestrategyofphagesurviving;**Cisactingelements:approximatetoagene,onthesameDNAstrand,regulategeneexpression.Anytranselements?Hoefaritshouldbe?**½DNAstrandsreleasegeneticinformationtomRNAandprotein;**Positiveandnegativecontrolofgeneexpression;**Genestructure:gene,ORF,CD,intronandexon,centraldogma.**DNAcloningvectorelements:cI,PLpromoter;**Genesplicing,Operon,transcribedunit,genecluster.**BAC,Bacterialartificialchromosome;cos,pAandothers.**Selfregulation(autoregulation)TP--OGene?P启动子突变后，引入野生型启动子，不能恢复基因转录，启动子与基因是一个顺反子，一个功能单位，一个基因。基因究竟如何界定？BacterialchromosomePlasmid/chromosomefragment你想过这个问题吗？Partialdiploid没有人刻意去划定基因在DNA上的界限

顺式调控元件与基因是一个行使功能的单位，它们是基因的一部分？内含子是否算基因的一部分？ORF是基因吗？不是的话，为什么在操纵子上那样划定？是的话，为什么mRNA上有非翻译区之说？mRNA对应基因吗？转录并不从P的起点开始。如果将顺式调控元件算基因的一部分，可以共用吗?知道了cDNA序列就克隆了基因吗？知道了序列就知道了基因吗？……。目前是一碗馄饨。问题是：你是明白人吗？行使单位功能需要的DNA上的所有的大大小小的片段以及这些片段之间的作用尚不明的大大小小的片段总和，有些段可以共享多次。问题是这些片段有哪些？你能下个定义吗？GeneStructure/基因界（限）定（义）

基因的认识或者定义过程就是分子生物学史Geneisdefinedwithstructureandunitfunction.

GeneislocatedonDNAonly.Itisasetofsequenceboxesfunctioningcooperativelyasaunitandendowinganorganismaunitcharacter.5‘5‘3‘3‘DNA一般是反向平行的两条链，B构型，结构参数，只要是连续的，就是DNA，长到染色体，短到几个、几十个碱基对，环状是连续的一种特殊形式。两条靠氢健结合的连续结构叫DNA的链，重链/轻链；编码链/摸板链，有意链/反意链，正链/付链；记录DNA时，用正/有意/编码链，一般是5‘-3’方向。DNA的分子量用长度表示，单位是碱基对(bp)、千碱基对(kb)、百万(兆)碱基对(Mb)、十亿碱基对(Gb)等。同一处，如果一条链上有磷酸二脂键断裂，叫nick，缺少一到数个碱基础，叫gap。末端用3‘的位置描述，3’protruding,3’recessive.限制性内切酶（以后讲）产生coherent末端，可以被连接酶连接起来。5‘位置上的磷酸基可以没去掉或加上，磷酸化酶/激酶，5’3‘都可以没接上其它生化基团。在DNA的复制（体内/体外）（核苷酸的任何一部分能为同位素/化学修饰过的类似物取代。DNA有变性/复性，既可以DNA/DNA，也能DNA/RNA，DNA复性只要60%以上的同源性，DNA复性与链组成/浓度/时间有关，有协同效应。我们已经认识的是低拷贝（基因组中），有基因的那些区域（长DNA），基因所在片段。更多？5‘3‘5‘3‘TemplatestrandCodingstrandRNApolmRNAinsitutranslationproteinIcTcORFopTRBSRBSSignalpGeneGenesinanoperonshareregulatingelementsmRNA全长是基因范围吗？如何看待Operon的问题？共享顺式调控元件？基因一定连续吗？基因内一定连续吗？内含子是否是基因的一部分？知道RNA就知道了基因吗？重叠基因算几个基因？AUGATGCodonsareonthemRNAonlyORF,onmRNA,fromstartingcodontostopcodon/oritscounterpartoncodingstrandofDNA(ourconvention,notnature);Openmeansunderstandable,byusandbycell;Understandablemeanswecandeduceaminoacidsequenceandcellcansynthesizeproteinfromit.TAATGATADOpenreadingframeORFATGATGNTAAMetTGNATGTGAProtein2fromthesamegenefragmentOverlappinggeneRBSshouldhaveuniquedesign,letting2possibilitiescoexistexon1exon2exon3intronintronFunctional?Yes!Enrichyourself.transcriptionsplicingcappingandtailingSpliceosome,oneoftwotypesFunctioning?算基因的结构成分？经常说基因有几个内含子。我们同时用断裂基因说明这样的情况。有时候，内含子有明确功能，如何界定？mRNA序列代表基因序列吗？RibosomalRNAgene(S)Transcriptionunit