分子生物學全

KillingMessengersDoublestrandedRNAmediatesthesilencingofthetranslationofitscorrespondingmRNA楊官品中國海洋大學海洋生命學院電話-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

（表達調(diào)控單元及其表達調(diào)控）一個Operon本來是可以為RNA聚合酶轉(zhuǎn)錄表達的，但因為Repressor的存在而不能轉(zhuǎn)錄了，這樣的調(diào)控形式稱負控制，從有到無的控制。Fromworkabletounworkable,negativecontrol.仍然是以operon的表達為落腳點，在其他都正常的情況下，某小分子的存在使負控制的Operon表達，稱可誘導的負控制(Induciblenegativecontrol)。誘導物不是乳糖Lactose，而是半乳糖苷Galactose。因為控制有泄漏Leakage，因此，半乳糖苷酶有衡量Traceamount表達，只要有乳糖存在，就能合成足以解除負控制的半乳糖苷。I基因編碼Repressor，組成型表達，基因可以與O/P臨近，也可以空間上分離；控制者元件Operator和促進者Promotor元件可以相臨，部分重疊或者完全重疊或空間上分割。*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(表達變不表達).Negativecontrolprovidesafail-safemechanism:iftheregulatorproteinisinactivated,thesystemfunctionsandsothecellisnotdeprivedoftheseenzymes.Forgenesunderpositivecontrol,expressionispossibleonlywhenanactiveregulatorproteinpresents(不表達變表達).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.

**被調(diào)控單元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.基因組最小接管一切細胞資源，如何做到？誰聰明，誰進化上更高級？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?整合效率遠遠低于復制可逆的，在沒有阻抑蛋白時，一個的整和不等于其它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、基因表達調(diào)控元件在DNA上，包括兩條DNA單鏈；基因表達調(diào)控元件與基因在同一DNA分子上，稱cis-elements;基因工程（半、準）中，一些基因或元件（如cI,P/O,T等）已被運用；Template,antisensestrand/coding,sensestrand;Heavy/lightstrand;基因?qū)懛?，coding/sensestrand,與mRNA共線性，基因在DNA上；DNA復制時，若另一鏈是連續(xù)合成的，遺傳信息多（做模板機會多，少突變機會），對滾環(huán)復制而言，不具有統(tǒng)計意義；溶原/裂解途徑的建立依賴PcII-PcIII調(diào)節(jié)蛋白組作為或者不作為；PcII-PcIII調(diào)節(jié)蛋白組作為或者不作為又依賴宿主細菌是否讓其作為；宿主細菌是否讓其作為依賴宿主細菌的基因型和生理狀態(tài)；當無作為時，…，Pcro關(guān)閉所有極端早期、延緩早期和cI基因轉(zhuǎn)錄，進入復制后，后期轉(zhuǎn)錄停止，復制裂解相關(guān)蛋白夠了，溶原相關(guān)蛋白也夠了，因為整合涉及底物、切與連等多步驟、低濃度，時間長或者機會少，因此，復制、裂解優(yōu)先；機會少不等于不發(fā)生，因此，具體到一個噬菌體，誰也說不準具體去向，這里的結(jié)論只是統(tǒng)計意義上的結(jié)論。當有作為時，…，因為在轉(zhuǎn)錄物的前面，應(yīng)該有多的機會獲得足夠的量先發(fā)生作用。發(fā)生作用時，先建立阻抑物蛋白的表達，然后自主調(diào)控，不斷表達，在復制、裂解相關(guān)蛋白足夠之前、后期轉(zhuǎn)錄開始或者未開始之前，有能力關(guān)閉所有的轉(zhuǎn)錄，即使后期有，也很快因PQ減少而停止；在生死攸關(guān)的時候，整合體系在不足夠的情況下，可以通過時間的延長和復制系統(tǒng)不作用時容許這樣的等待存在的情況下，總有機會整合進宿主染色體，進入溶原狀態(tài)，一個具體的噬菌體也可能由此消失（生物稀釋作用）。這里說的仍然是統(tǒng)計意義上的結(jié)論。有作為時，就開始用反義RNA的形式蹜蹜減少PcII和Pcro；因為只要啟動了PRE,進一步的轉(zhuǎn)錄就不必要了，解除Pcro可以解除對PRM的限制，Pcro對左右轉(zhuǎn)錄的限制，可以有PcI取代；唯一DNA的兩條鏈都被使用的例子，人為的使用例子很多；cI自主調(diào)控能成立時，PRE早就不需要了；RNAPol雙向等機會；對PcI能發(fā)揮作用的噬菌體，同種或含有相同調(diào)節(jié)蛋白的，具有免役作用；只要解除PcI的作用，就能重新啟動轉(zhuǎn)錄，面臨重新決定，因為PcI的作用都能解除，PcII/PcIII不作為的機會多，但具體到一個噬菌體，誰也說不準；機會少，但一旦有作為，就重新維持溶原狀態(tài)；一旦無作為，復制裂解、重組整合的相關(guān)蛋白都足夠，等在那里耗損時間，一旦切下來，立即進入復制裂解周期。**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.調(diào)節(jié)蛋白/負控制/通過降解實現(xiàn)變構(gòu)/導致原因不存在？表達？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可阻斷正控制Pcroisarepressorneededforlyticinfection**ItinhibitstheexpressionofearlygenesfrombothPLandPR;**TheaffinityofpcrotoPR3isgreaterthanthattoOR2orOR1.ItinhibitsRNApolfrombindingtoPRM(itsfirstaction);**pcrobindstoOR2orOR1.Itsaffinityforthesetwositesissimilar.Thereisnocooperativeeffect.**ItspresenceateithersiteissufficienttopreventRNApolfrombindingfrombothpromoters;Thisinturnstopstheproductionoftheearlyfunction,includingpcroitself(itssecondaction);**BecauseCIIisunstable(如果穩(wěn)定就是另一種情況),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?**?DNAstrandsreleasegeneticinformationtomRNAandprotein;**Positiveandnegativecontrolofgeneexpression;**Genestructure:gene,ORF,CD,intronandexon,centraldogma.**DNAcloningvectorelements:cI,PLpromoter;**Genesplicing,Operon,transcribedunit,genecluster.**BAC,Bacterialartificialchromosome;cos,pAandothers.**Selfregulation(autoregulation)TP--OGene?P啟動子突變后，引入野生型啟動子，不能恢復基因轉(zhuǎn)錄，啟動子與基因是一個順反子，一個功能單位，一個基因?；蚓烤谷绾谓缍?？BacterialchromosomePlasmid/chromosomefragment你想過這個問題嗎？Partialdiploid沒有人刻意去劃定基因在DNA上的界限

順式調(diào)控元件與基因是一個行使功能的單位，它們是基因的一部分？內(nèi)含子是否算基因的一部分？ORF是基因嗎？不是的話，為什么在操縱子上那樣劃定？是的話，為什么mRNA上有非翻譯區(qū)之說？mRNA對應(yīng)基因嗎？轉(zhuǎn)錄并不從P的起點開始。如果將順式調(diào)控元件算基因的一部分，可以共用嗎?知道了cDNA序列就克隆了基因嗎？知道了序列就知道了基因嗎？……。目前是一碗餛飩。問題是：你是明白人嗎？行使單位功能需要的DNA上的所有的大大小小的片段以及這些片段之間的作用尚不明的大大小小的片段總和，有些段可以共享多次。問題是這些片段有哪些？你能下個定義嗎？GeneStructure/基因界（限）定（義）

基因的認識或者定義過程就是分子生物學史Geneisdefinedwithstructureandunitfunction.

GeneislocatedonDNAonly.Itisasetofsequenceboxesfunctioningcooperativelyasaunitandendowinganorganismaunitcharacter.5‘5‘3‘3‘DNA一般是反向平行的兩條鏈，B構(gòu)型，結(jié)構(gòu)參數(shù)，只要是連續(xù)的，就是DNA，長到染色體，短到幾個、幾十個堿基對，環(huán)狀是連續(xù)的一種特殊形式。兩條靠氫健結(jié)合的連續(xù)結(jié)構(gòu)叫DNA的鏈，重鏈/輕鏈；編碼鏈/摸板鏈，有意鏈/反意鏈，正鏈/付鏈；記錄DNA時，用正/有意/編碼鏈，一般是5‘-3’方向。DNA的分子量用長度表示，單位是堿基對(bp)、千堿基對(kb)、百萬(兆)堿基對(Mb)、十億堿基對(Gb)等。同一處，如果一條鏈上有磷酸二脂鍵斷裂，叫nick，缺少一到數(shù)個堿基礎(chǔ)，叫g(shù)ap。末端用3‘的位置描述，3’protruding,3’recessive.限制性內(nèi)切酶（以后講）產(chǎn)生coherent末端，可以被連接酶連接起來。5‘位置上的磷酸基可以沒去掉或加上，磷酸化酶/激酶，5’3‘都可以沒接上其它生化基團。在DNA的復制（體內(nèi)/體外）（核苷酸的任何一部分能為同位素/化學修飾過的類似物取代。DNA有變性/復性，既可以DNA/DNA，也能DNA/RNA，DNA復性只要60%以上的同源性，DNA復性與鏈組成/濃度/時間有關(guān)，有協(xié)同效應(yīng)。我們已經(jīng)認識的是低拷貝（基因組中），有基因的那些區(qū)域（長DNA），基因所在片段。更多？5‘3‘5‘3‘TemplatestrandCodingstrandRNApolmRNAinsitutranslationproteinIcTcORFopTRBSRBSSignalpGeneGenesinanoperonshareregulatingelementsmRNA全長是基因范圍嗎？如何看待Operon的問題？共享順式調(diào)控元件？基因一定連續(xù)嗎？基因內(nèi)一定連續(xù)嗎？內(nèi)含子是否是基因的一部分？知道RNA就知道了基因嗎？重疊基因算幾個基因？AUGATGCodonsareonthemRNAonlyORF,onmRNA,fromstartingcodontostopcodon/oritscounterpartoncodingstrandofDNA(ourconvention,notnature);Openmeansunderstandable,byusandbycell;Understandablemeanswecandeduceaminoacidsequenceandcellcansynthesizeproteinfromit.TAATGATADOpenreadingframeORFATGATGNTAAMetTGNATGTGAProtein2fromthesamegenefragmentOverlappinggeneRBSshouldhaveuniquedesign,letting2possibilitiescoexistexon1exon2exon3intronintronFunctional?Yes!Enrichyourself.transcriptionsplicingcappingandtailingSpliceosome,oneoftwotypesFunctioning?算基因的結(jié)構(gòu)成分？經(jīng)常說基因有幾個內(nèi)含子。我們同時用斷裂基因說明這樣的情況。有時候，內(nèi)含子有明確功能，如何界定？mRNA序列代表基因序列嗎？RibosomalRNAgene(S)Transcriptionunit