分子生物学（杨洋）课程总结-

TherevisedcentraldogmaTranslationRNAprocessingDNArepair基因组的保持基因组的表达regulation分子生物学技术（原理及应用）课程总结第一章-绪论，DNA和RNA结构第二章-染色体，染色质和核小体(Chromosomes,chromatin,andthenucleosome）第三章-DNA的复制(ThereplicationofDNA）第四章-DNA的突变和修复(ThemutabilityandrepairofDNA）第五章-转录机制(MechanismsofTranscription)第六章RNA剪接(RNASplicing）第七章翻译-1，2,3第八章-遗传密码(geneticcode)第九章-分子生物学技术(Techniquesofmolecularbiology)第十章原核生物基因表达调控(Regulationinprokaryotes)第十一章真核生物基因表达调控(Regulationineukaryotes)第十二章-DNA重组第一章-绪论，DNA和RNA结构分子生物学的含义分子生物学的发展分子生物学研究内容WhatisMolecularbiology?

Defineinbroadly:（广义定义）understandbiologicalphenomenainmolecularterms(difficulttodistinguishfrombiochemistry)Defineinrestrictively:（狭义定义）thestudyofgenestructureandtheiractivitiesinmolecularlevel（分子水平上研究基因的结构和功能）GeneconceptGenestructureGenereplicationGeneexpressionGenerecombinationGenemutationMolecularBiologyofGene（基因的分子生物学）第二章-染色体，染色质和核小体(Chromosomes,chromatin,andthenucleosome）Chromosomesequence&diversity

（染色体序列和多样性）Thenucleosome（核小体）Higher-orderchromatinstructure

（染色质的高级结构）Regulationofchromatinstructure

（染色质结构的调控）Nucleosomesarethebuildingblocksofchromosomes（核小体是染色体的结构单位）在真核细胞中大多数DNA被包装进核小体ThenucleosomeiscomposedofacoreofeighthistoneproteinsandtheDNA(coreDNA,147bp，核心DNA)wrappedaroundthem（核小体由8个组蛋白所形成的核组成，DNA缠绕在组蛋白核上）TheDNAbetweeneachnucleosomeiscalledalinkerDNA（连接DNA).EacheukaryotehasacharacteristicaveragelinkerDNAlength(20-60bp)Figure7-18DNApackagedintonucleosomeSix-foldDNAcompactionDNA盘绕在组蛋白核上（H2A,H2B,H3，H4）第三章-DNA的复制(ThereplicationofDNA）1.TheChemistryofDNASynthesis(DNA合成的化学基础）2.TheMechanismofDNAPolymerase（DNA聚合酶的作用机制）3.TheSpecializationofDNAPolymerases（DNA聚合酶的特化）4.TheReplicationFork（复制叉）5.DNASynthesisattheReplicationFork（复制叉上的DNA合成）6.InitiationofDNAReplication（复制的起始）7.BindingandUnwinding（结合和解旋）8.FinishingReplication（复制的终止）TheChemistryofDNASynthesis:substrate,directionandenergy.TheMechanismofDNAPolymerase:1polymerizationmechanism,2differentwaysofdiscriminatingsubstrates,2catalyticsites;3domains.TheSpecializationofDNAPolymerasesTheReplicationFork:theenzyme/proteinsrequiredtosynthesizetheleadingandlaggingstrands.DNASynthesisattheReplicationFork:Holoenzyme/trombonemodeltoexplainhowtheanti-paralleltemplatestrandsarecopied/replicatedtowardthereplicationfork.Replisome/proteininteraction.SummaryInitiationofDNAReplication/bindingandunwinding:therepliconmodel;initiationinbacteria;initiationcontrolineukaryotes-alinkwithcellcycle(pre-RCassemblyandactivation).FinishingReplication:Telomeres,telomerasePCR（聚合酶链式反应）

Polymerasechainreaction

AnimportanttechniquebasedonDNAPolymerase

Thepolymerasechainreaction(PCR)istousedtoamplifyasequenceofDNAinvitro,usingapairofprimerseachcomplementarytooneendofthetheDNAtargetsequence.复制理论实验技术Denaturation(变性):ThetargetDNA(template)isseparatedintotwostandsbyheatingto95℃Primerannealing(退火):Thetemperatureisreducedtoaround55℃toallowtheprimerstoanneal.Polymerization(elongation,extension)(延伸):Thetemperatureisincreasedto72℃foroptimalpolymerizationstepwhichusesdNTPsandrequiresMg++.TheprincipleofPCR:ThreedifferentstepsproceedineachPCRcycle.Reversetranscriptase(RT)-PCR逆转录PCRAAA(A)n5‘-CapmRNA(dT)12~18

primeranneal5‘-CapAAA(A)n3‘5‘ReversetranscriptiondNTP,RT5‘-CapAAA(A)n5‘cDNA:mRNAhybridRegularPCR第四章-DNA的突变和修复(ThemutabilityandrepairofDNA）Replicationerrorsandtheirrepair(复制错误及其修复）DNAdamage（DNA损伤）RepairofDNAdamage（DNA损伤的修复）复制中产生的错误以及它们是如何被修复的自发产生或来自外界的攻击造成的各种损伤细胞修复损伤的多种修复机制生物在保持遗传物质忠实性方面的努力复制中产生的错误以及它们是如何被修复的自发产生或来自外界的攻击造成的各种损伤—DNA损伤的各种类型细胞修复DNA损伤的多种修复机制：1.DirectreversalofDNAdamagebyphotoreactivation(光活化作用)andalkyltransferase(烷基转移酶)-直接将损伤逆转2.剪切修复系统（excisionrepairsystem)：仅仅将受损的核苷酸去除将包含损伤的一小段单链DNA去除3.重组修复系统（Recombinationrepair）：当DNA两条链都受损（断裂）时采用这种修复—双链断裂修复（Double-strandbreakrepair）4.移损DNA合成（TranslesionDNAsynthesis）：DNA聚合酶的复制进程被受损碱基阻碍时采用的修复1.DirectreversalofDNAdamagebyphotoreactivation(光活化作用)andalkyltransferase(烷基转移酶)-直接将损伤逆转2.剪切修复系统（excisionrepairsystem)：另一条未受损的链作为模板，以便DNA聚合酶重新掺入正确的核苷酸仅仅将受损的核苷酸去除将包含损伤的一小段单链DNA去除3.重组修复系统（Recombinationrepair）：当DNA两条链都受损（断裂）时采用这种修复—双链断裂修复（Double-strandbreakrepair）4.移损DNA合成（TranslesionDNAsynthesis）：DNA聚合酶的复制进程被受损碱基阻碍时采用的修复Mechanismstorepairadamage（DNA损伤的修复系统）第五章-转录机制(MechanismsofTranscription)RNApolymeraseandthetranscriptioncycle（RNA聚合酶和转录周期）Thetranscriptioncycleinbacteria（细菌的转录周期）Transcriptionineukaryotes（真核生物的转录）RNApolymerases(RNAP,真核和原核的异同)andtranscriptioncycle(Initiation,elongationandtermination)Transcriptioncycleinbacteria:-Initiation:(1)Thefeatureofs70promoters.(2)Promoterbindingbys70transcriptionfactor(recognitionmechanism).(3)Transitiontoopencomplex.(4)Promoterescapeandtransitiontotheternarycomplex.-Elongationandeditingbypolymerase-Termination:Rho-independentandRho-dependentmechanism.TranscriptionineukaryotesbyRNAPII:

Initiation

(i)Cis-actingelements:corepromoter®ulatorysequences (ii)Formationofthepre-initiationcomplex (iii)PromoterescapeandtheCTDtail (iv)ThefunctionofeachGTF(invitro) (v)Additionalproteinsforinvivotranscription.ElongationandproofreadinginvolveanewsetofGTFs

CoupledwithRNAprocessing5’cappingand3’polyadenylation(mechanisms)Termination4.TranscriptionineukaryotesbyRNAPIandIII-initiation:Promoterbindingandformationoftheclosedcomplex.第六章RNA剪接(RNASplicing）

Figure13-1Primarytranscript外显子内含子前mRNA剪接后的mRNARNA剪接的化学基础剪接体剪接过程可变剪接自剪接内含子RNA编辑mRNA转运WhyRNAsplicingisimportant?Chemicalreaction:determinationofthesplicesites,theproducts,trans-splicingSpliceosome:splicingpathwayandfindingthesplicesites.Self-splicingintronsandmechanismsAlternativesplicingandregulationTwodifferentmechanismsofRNAeditingmRNAtransport-alinktotranslation

第七章翻译-1，2,3

Topic1-4:Fourcomponentsoftranslationmachinery

mRNA

tRNAattachmentofaminoacidstotRNA(aminoacyl-tRNA

synthetases)ribosomeTopic5-7:Translationprocess

initiation;elongation;terminationTopic8:Antibiotics(抗生素）andtranslation5/1/202629BasicmachineryofTranslationmRNAs(~5%oftotalcellularRNA)-messageRNAtRNAs

(~15%)-transferRNAaminoacyl-tRNA

synthetases

(氨酰tRNA合成酶)ribosomes(核糖体):rRNAOverviewoftheeventsoftranslationTermination

Elongation

InitiationThreeeventsmustoccur:ribosomemustberecruitedtomRNA2.achargedtRNAmustbeplacedintothePsiteofribosome3.theribosomemustbepreciselypositionedoverthestartcodonThreeinitiationfactors(IF)directtheassemblyofaninitiationcomplexthatcontainsmRNAandinitiatortRNA

Translationinitiationfactors(IF):IF1:preventtRNAfrombindingtotheAsiteinthesmallsubunitIF2:aGTPasethatinteractswithsmallsubunit,IF1andinitiatortRNA(fMet-tRNAfMet);IF2canfacilitatetheassociationof

fMet-tRNAfMetwiththesmallsubunitIF3:bindstothesmallsubunitandblocksitfrom

reassociatingwithlargesubunit,helpstodissociatethe70Sribosomeintoitslargeandsmallsubunit进位易位肽键形成Twoclassesofreleasefactors

ClassI:recognizethestopcodonandtriggerhydrolysisofthepolypeptidefrompeptidyl-tRNA

Prokaryotes:RF1-UAG,UAARF2-UGA,UAAEukaryotes:eRF1-UAG,UGA,UAAClassII:stimulatethedissociationoftheclassIfactorsfromtheribosomeafterreleaseofpolypeptide

Prokaryotes:RF3Eukaryotes:eRF3GTP-bindingprotein

Antibiotics(抗生素）andtranslationPuromycin(嘌呤霉素)Puromycinresemblestheaminoacylated-tRNA,soitcanbindtotheAsiteofribosome

Puromycin(嘌呤霉素)肽酰嘌呤霉素(peptidyl-puromycin)Puromycin第八章-遗传密码(geneticcode)

1遗传密码的破译2遗传密码的特征与性质3遗传密码的简并性和摆动假说4改变遗传密码的几类突变

4.1错义突变

4.2无义突变

4.3移码突变5基因内抑制突变和基因间抑制突变6遗传密码的通用性“Thegeneticcodeisdegenerate”Whatdoesitmean?Whatarethebenefits?Whatisthewobbleconcept?HowthewobbleintheanticodonaffectthenumberoftRNAstorecognizethe61codons?Whatarethemutationsalteringgeneticcode?Whataresuppressormutations?Whatisthedifferencebetweenintragenicsuppressionandintergenicsuppression?Whatarethebenefitsofthecodeuniversality?

IntergenicSuppressionInvolvesmutanttRNAsMutanttRNAgenessuppresstheeffectsofnonsensemutationsinprotein-codinggenes.Theyactbyreadingastopcodonasifitwereasignalforaspecificaminoacid.第九章-分子生物学技术(Techniquesofmolecularbiology)

NucleicacidsTechniquesProteinTechniquesTechniquesforstudyingDNA-proteininteractionTechniquesforstudyingprotein-proteininteractionBasicprincipleBasicprocedureapplicationNucleicacidsTechniques（核酸技术）SeparationbyElectrophoresis(电泳分离)CutbyRestrictionendonuclease(限制性内切酶切割技术)DNACloningandgeneexpression(基因克隆和表达技术）分子杂交技术（DNA杂交技术，RNA杂交技术）-Southern

hybridization,Northern

hybridization,WesternhybridizationPCR技术（聚合酶链式反应）Restrictiondigestionofyourinsertandvectorusingthesameenzyme.Useligase(连接酶）tojoinyourinsertandvectortogether.Transformtheligationproducts

intoE.colicompetentcells.Selectthedesiredclones:

Growthecellsonaplatecontainingtetracycline(四环素).EcoRIProteinTechniques(蛋白质技术）Proteinpurification(蛋白质纯化)Affinitychromatographycanfacilitatemorerapidproteinpurification(亲和层析纯化)ProteinseparationbyPAGEgelelectrophoresis(蛋白质分离)andidentificationbyWesternanalysis(免疫印迹)

（一）DNA-proteinaffinitychromatography(DNA-蛋白亲和层析)(二）Gelretardation(凝胶阻滞,EMSA，竞争性EMSA)（三）DNaseIfootprinting(DNaseI足迹法)（四）染色质免疫共沉淀技术

（chromatinimmunoprecipitationassay,CHIP）

-研究体内DNA与蛋白质相互作用的方法

（五）Yeastone-hybridsystem(酵母单杂交)

StudytheinteractionbetweenproteinandnucleicacidTechniquesforstudying

protein-proteininteractionYeasttwo-hybridsystem(酵母双杂交)

免疫共沉淀技术targetbaitNotranscriptiontargetbaitFishingwiththeyeasttwo-hybridsystembait

targetdoesXbindwithaunknownprotein?proteinXbaitBDADBDBDcDNA

library+ADfusionplasmid

transformLibrariesofDNAmoleculescanbecreatedbycloning(GenomiclibraryandcDNAlibrary)ADNAlibrary(DNA文库)isapopulationofidenticalvectorsthateachcontainsadifferentDNAinsert.GenomicLibrary(基因组文库):theDNAinsertsinaDNAlibraryisderivedfromrestrictiondigestionofthegenomicDNA.cDNAlibrary(cDNA文库):theDNAinsertsinaDNAlibraryisconvertedfromthemRNAsofatissue,acelltypeoranorganism.cDNAstandsfortheDNAcopiedfrommRNA.

Proteomics(蛋白质组学)Threeprinciplemethods1.2-Dgelelectrophoresisforproteinseparation(蛋白质分离).2.MSspectrometryfortheprecisedeterminationofthemolecularweightandidentifyofaprotein(蛋白质鉴定).3.Bioinformaticsforassigningproteinsandpeptidestothepredictedproteincodingsequenceinthegenome(蛋白质确定).原理机制应用RNA干扰技术

（RNAinterference,RNAi）

第十章原核生物基因表达调控(Regulationinprokaryotes)

TOPIC1PrinciplesofTranscriptionalRegulationTOPIC2RegulationofTranscriptionInitiation:ExamplesfromBacteria(Lacoperon,乳糖操纵子)TOPIC3ExamplesofGeneRegulationafterTranscriptionInitiation(Trp

operon，色氨酸操纵子)Topic4Majorshiftsinprokaryotictranscription-Alternativeσfactor

（基于可变

因子的全局性调控）TOPIC1PrinciplesofTranscriptionalRegulationGeneexpressioniscontrolledatdifferentstages(基因表达调控可以发生在不同时期):ThekeystepofgeneregulationtakesplaceattheinitiationoftranscriptionGeneExpressionisControlledbyRegulatoryProteins(调控蛋白)PositiveregulatorsoractivatorsNegativeregulatorsorrepressorsrecruitmentregulation(招募调控)Allosteryregulation(异构调控):convertstheclosedcomplextoopencomplex

Basicprincipleofregulationofgeneexpression:Interactionbetweentrans-actingfactor(regulatoryprotein)andcis-actingelement(controlelement):反式作用因子和顺式作用元件

ControlelementStructuralgenesBasicprincipleofregulationofgeneexpression:Interactionbetweentrans-actingfactor

(regulatoryprotein)andcis-actingelement

(controlelement)反式作用因子和顺式作用元件之间的相互作用trans-actingfactorTOPIC2RegulationofTranscriptionInitiation:ExamplesfromBacteria(Lacoperon)Operon(操纵子,操纵元)StructuralgenesControlelements,suchasoperator(操作子)sequenceRegulatorgeneOperon(操纵子,操纵元):

aunitofprokarytoicgeneexpressionandregulationwhichtypicallyincludes:1.Structuralgenesforenzymesinaspecificbiosyntheticpathwaywhoseexpressioniscoordinatelycontrolled

2.Controlelements,suchasoperator(操作子)sequence-cis-actingelement

3.Regulatorgene(s)codeforregulatoryproteinwhichcanrecognizeandinteractwiththecontrolelementsNegativecontroloftheLac

operon:interactionbetweenrepressorandoperator(乳糖操纵子负调控)Twokeyelements:

lacI:encodinglacrepressor,trans-actingfactoroperator:lac

repressorbindingsite,cis-actingelementlacIpromoterRNApolymeraselacZlacYlacANotranscriptiontranscriptionoperatoractiverepressorinactiverepressor阻遏去阻遏PositivecontroloftheLac

operon:interactionbetweenCAP-cAMPcomplexandCAPbindingsite(乳糖操纵子正调控)CAP(CataboliteActivatorProtein,代谢产物激活蛋白)orCRP(cAMPReceptorProtein,cAMP受体蛋白)theCAP-cAMPbindingsiteMechanismCAP-cAMPCAPbindingsitelacZTranscription（转录）促进了RNA聚合酶和启动子结合形成稳定的开放复合体(RNA聚合酶)(启动子)TheLACoperon

TOPIC3ExamplesofGeneRegulationafterTranscriptionInitiation(Trp

operon)Twolayersofregulationareinvolved:

(1)transcriptionrepressionbytheTrprepressor(阻遏作用)-粗调

(2)attenuation(衰减作用)-细调

TranscriptionrepressionbytheTrprepressor(阻遏作用)-粗调低色氨酸:去阻遏高色氨酸:阻遏inactiverepressoractiverepressorNotranscription色氨酸14个氨基酸的前导肽LowTrpHighTrpFig16-21Complementary2:3ElongationoftranscriptionComplementary3:4terminationoftranscriptionTopic4:

Majorshiftsinprokaryotictranscription-Alternativeσfactor

（基于可变

因子的全局性调控）

细菌经历逆境：饥饿，热激，缺氮；枯草芽孢杆菌的孢子形成等细菌通过转录的全局性变化对其环境变化作出应答，这些全局性的转录变化是通过RNA聚合酶的变化得以实现的Holoenzyme=factor+coreenzyme

RNA聚合酶的变化本质上是σ因子的变化，

σ因子控制转录的特异性细菌通过转录的全局性变化对其环境变化作出应答，这些全局性的转录变化是通过RNA聚合酶的变化得以实现的第十一章真核生物基因表达调控(Regulationineukaryotes)

TOPIC1:PrinciplesofRegulationineukaryotesTopic2:TranscriptionfactorsTopic3:chromatinstructureanditseffectontranscriptionTopic4:eukaryoticrepressorsTOPIC1PrinciplesofRegulationineukaryotesSimilarityofregulationbetweeneukaryotesandprokaryoteDifferenceinregulationbetweeneukaryotesandprokaryoteDifferenceinregulationbetweeneukaryotesandprokaryotePre-mRNAsplicingaddsanimportantstepforregulation.(intron,extron,mRNA前体的剪接)Theeukaryotictranscriptionalmachineryismoreelaboratethanitsbacterialcounterpart.(真核转录机器更复杂,transcriptionfactors)Nucleosomesandtheirmodifiersinfluenceaccesstogenes.(核小体及其修饰体)Manyeukaryoticgeneshavemoreregulatorybindingsitesandarecontrolledbymoreregulatoryproteinsthanarebacterialgenes.(真核基因有更多调控蛋白结合位点)

TOPIC2TranscriptionfactorsGeneraltranscriptionfactors:BasalleveltranscriptionGene-specifictranscriptionfactors(activator)Eukaryoticactivators(真核激活蛋白)haveseparateDNAbindingandactivatingfunctions:containsseparateDNAbindingandactivatingdomainsThetwohybridAssay(酵母双杂交)basedontheseparationofDNAbindingandactivatingdomainsisusedtoidentifyproteinsinteractingwitheachotherEukaryoticactivators(真核激活蛋白)alsoworkbyrecruiting(招募)asinbacteria

Eukaryoticactivatorsrecruitpolymeraseindirectlyintwoways:1.Interactingwithpartsofthe

transcriptionmachineryexceptforRNApolymerase.2.Recruitingnucleosomemodifiersthatalterchromatininthevicinityofagene.Theeukaryotictranscriptionalmachinerycontainspolymeraseandnumerousproteinsbeingorganizedtoseveralcomplexes,suchastheMediatorandtheTFⅡDcomplex.Activatorsinteractwithoneormoreofthesecomplexesandrecruitthemtothepromoter.Figure17-9Topic3:chromatinstructureanditseffectontranscriptionNucleosomesarethebuildingblocksofchromosomesHistonesaresmall,positivelycharged(basic)proteinsThecorehistoneseachhaveanN-terminal“tail”,thesitesofextensivemodificationsHistonenotonlyisthestructuralcomponentofnucleosome,butalsocanregulatethegeneexpressionhistonemodificationsuchasacetylation（组蛋白乙酰化酶）

Topic4:eukaryoticrepressorsIneukaryotes,mostrepressorsdonotrepresstranscriptionbybindingtositesthatoverlapwiththepromoterandthusblockbindingofpolymerase.(Bacteriaoftendoso)Commonly,eukaryoticrepressorsrecruitnucleosomemodifiersthatcompactthenucleosomeorremovethegroupsrecognizedbythetranscriptionalmachineryContrasttotheactivatorrecruitednucleosomemodifers,histonedeacetylases(组蛋白去乙酰化酶)repressthetranscriptionbyremovingtheacetylgroupsSomemodifieraddsmethylgroupstothehistonetails,whichfrequentlyrepressthetranscription.Thismodificationcausestranscriptionalsilencing.Threeotherwaysinwhichaneukaryoticrepressorworksinclude:Competeswiththeactivatorforanoverlappedbindingsite.Bindstoasitedifferentfromthatoftheactivator,butphysicallyinteractswithanactivatorandthusblockitsactivatingregion.Bindstoasiteupstreamofthepromoter,physicallyinteractswiththetranscriptionmachineryatthepromotertoinhibittranscriptioninitiation.Figure17-19：WaysinwhicheukaryoticrepressorworkCompetesfortheactivatorbindingInhibitsthefunctionoftheactivator.BindstothetranscriptionmachineryRecruitsnucleosomemodifiers(mostcommon)第十二章-DNA重组ModelsforHomologousRecombination（同源重组的两种模式）HomologousRecombinationProteinMachines(RecBCDpathway)（同源重组的蛋白质机制）HomologousRecombinationinEukaryotes（真核细胞的同源重组）GeneticConsequencesoftheMechanismofHomologousRecombination(exchangeorconversion)（同源重组的遗传结果）TherevisedcentraldogmaTranslationRNAprocessingDNArepair基因组的保持基因组的表达regulation分子生物学技术（原理及应用）杨洋教授研究领域：微生物分子生物学和微生物遗传学目前主要研究领域为白腐真菌及其漆酶的分子生物学和遗传学：白腐真菌参与生物质和异生物质降解和转化的重要功能基因的克隆与分析白腐真菌降解相关基因的表达调控分子机制的研究白腐真菌高效降解生物质与异生物质的分子作用机理研究高效降解环境污染物的基因工程菌的构建与利用及高效降解酶的定向分子进化研究详细介绍请参阅:8181/menu020004/6419.jhtml

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