分子生物学（杨洋）第七章 翻译-1

2026/5/41WelcometoMyMolecularBiologyClass

YangYang（杨洋教授）

HuazhongUniversityofSciencesandTechnology2026/5/42MolecularBiologyoftheGene,6/E

---Watsonetal.(2008)PartI:ChemistryandGeneticsPartII:MaintenanceoftheGenomePartIII:ExpressionoftheGenomePartIV:RegulationPartV:Methods2026/5/43PartIII:ExpressionoftheGenomeThispartconcernedwithoneofthegreatestchallengesinunderstandingthegene－howthegeneisexpressed2026/5/44Ch12:Mechanismsoftranscription

Ch13:RNAsplicingCh14:TranslationCh15:ThegeneticcodeCHAPTER7Translation（第七章翻译）2026/5/46TherevisedcentraldogmaTranslationRNAprocessingDNArepairandrecombination基因组的保持基因组的表达2026/5/47Whatistranslation?

--itisthestoryaboutdecodingthegeneticinformationcontainedinmessengerRNA(mRNA)intoproteinsgeneticinformation:nucleotidesinmessengerRNA(mRNA)

protein:linearsequencesofaminoacidsinprotein

2026/5/48ThelanguageofmRNA:fouralphabet(A,G,C,U)Thelanguageofprotein:twentyalphabet(aminoacid)translation2026/5/49DNA：5-ATGAGTAACGCG-33-TACTCATTGCGC-5NontemplatestrandtemplatestrandtranscriptionmRNA：5-AUGAGUAACGCG-3translationProtein：Met-Ser-Asn-Ala2026/5/410TranslationextremelycostsInrapidgrowingbacterialcells,proteinsynthesisconsumes80%ofthecell’senergy50%ofthecell’sdryweightWhy?2026/5/411OutlineTopic1-4:Fourcomponentsoftranslationmachinery

mRNAtRNAattachmentofaminoacidstotRNA(aminoacyl-tRNAsynthetases)ribosomeTopic5-7:Translationprocess

initiation;elongation;terminationTopic8:Antibiotics(抗生素）andtranslation2026/5/412BasicmachineryofTranslationmRNAs(~5%oftotalcellularRNA)-messageRNAtRNAs(~15%)-transferRNAaminoacyl-tRNAsynthetases(氨酰tRNA合成酶)ribosomes(核糖体):rRNA2026/5/413Whatisthekeyattributeofeachcomponent?Howthefourcomponentworktogethertoaccomplishtranslation?reductionism(还原论)holism(整体论)philosophy2026/5/414Questionsaddressedin

“BasicmachineryofTranslation”WhatistheorganizationofnucleotidesequenceinformationinmRNA?WhatisthestructureoftRNAs?HowdoaminoacyltRNAsynthetasesrecognizeandattachthecorrectaminoacidstoeachtRNA?Howdoestheribosomeorchestratethetranslationprocess?2026/5/415Topic1:mRNA(信使RNA)DNA3-nt-longcodons(三联体密码子)specifytheorderofaminoacidsmRNAtranscription2026/5/416DNA：5-ATGAGTAACGCG-33-TACTCATTGCGC-5NontemplatestrandtemplatestrandtranscriptionmRNA：5-AUGAGUAACGCG-3translationProtein：Met-Ser-Asn-Ala2026/5/4171-1polypeptidechainsarespecifiedbyORF

openreadingframe(ORF,开放阅读框):acontiguous,non-overlappingstringofcodonsEachORFspecifiesasingleproteinandbeginswithastartcodonandendswithastopcodon2026/5/418Thestartcodon（起始密码子）

—thefirstcodonofanORFInbacteria:AUG,GUG,orUUG(5’-3’)Ineukaryoticcells:5’-AUG-3’Functions:1.Specifiesthefirstaminoacidtobeincorporatedintothegrowingpolypeptidechain2.Definesthereadingframe

（阅读框）forallsubsequentcodons2026/5/419startcodon2026/5/420Thestopcodon（终止密码子）UAG,UGA,orUAA(5’-3’)Functions:1.DefinestheendofORF2.Signalterminationofpolypeptidesynthesis2026/5/421openreadingframe(ORF):

acontiguousstretchofcodons“read”inaparticularframe(decidedbystartcodon)

“open”totranslationbecauseitlacksastopcodon(untilthelastcodoninORF)2026/5/422EukaryoticmRNA(真核生物mRNA)monocistrionic,单顺反子polypeptidemRNA2026/5/423ProkaryoticmRNA(原核生物mRNA)polycistrionic,多顺反子Polypeptide-1Polypeptide-2Polypeptide-3Why?ORF1ORF2ORF32026/5/424EukaryoticmRNA(monocistrionic,单顺反子):onemRNAcontainsoneORFonemRNAProkaryoticmRNA(polycistrionic,多顺反子):onemRNAcontainsmultipleORFsonemRNAOneploypeptideMultiplepolypeptides(relatedfunction)2026/5/4251-2ProkaryoticmRNAshavearibosomebindingsite

(核糖体结合位点)thatrecruitsthetranslationalmachinery(核糖体)原核生物mRNA结构特点：2026/5/426Ribosomebindingsite(RBS)orSD-sequence:complementarywiththesequenceatthe3’endof16SrRNA.ORF1ORF2ORF32026/5/427Ribosome(核糖体):rRNA2026/5/428Translationalcoupling(翻译耦合)5’-AUGA-3’stopcodonstartcodonORF1ORF2ORF3overlappingFindingofRibosomebindingsite(RBS)orSD-sequenceScientific

question:howcanthecellrecognizethecorrectstartcodon?AUGAUGAUGstartcodonordinarycodonModel:R17phageinfectsE.coliR17phage:threegenes—codingforAprotein,coatproteinandreplicase(复制酶）Phenomenon:生物学现象E.coli

（大肠杆菌）ribosomecantranslateallthreephagegenesinvitro(体外）RibosomefromanotherbacteriumBacillus（芽孢杆菌）canonlytranslatetheAproteingeneWhy?Ifthe30SsubunitisfromE.coli,phagecoatproteingenecanbetranslatedIfthe30SsubunitisfromBacillus,phagecoatproteingenecannotbetranslatedExperimentalresults:30Ssubunit（核糖体30S

小亚基）isthekeyfactordeterminingthetranslation??30SsubunitwasfurtherdissociatedintoRNAandprotein

Ifthe16srRNAisfrom

E.coli,phagecoatproteingenecanbetranslatedIfthe16srRNAisfrom

Bacillus,phagecoatproteingenecannotbetranslated16srRNA

isthekeyfactordeterminingthetranslationORF1ORF2ORF3J.ShineandL.DalgarnofindtheSDsequence:MorepowerfulexperimentalevidencefortheimportanceofSD-sequence

SD:GGAGGHumangrowthhormonegenewildtypeE.colicell(16srRNA:CCUCC)Producemuchhumangrowthhormoneprotein

（人生长激素蛋白）SD:GUGUGHumangrowthhormonegeneProducenohumangrowthhormoneproteinwildtypeE.colicell(16srRNA:CCUCC)突变的研究思想SD:GUGUGHumangrowthhormonegeneProducemuchhumangrowthhormoneproteinmutatedtypeE.colicell

(16srRNA:CACAC)Phenomenon（现象）Discoveryofmechanism（机制）behindthisphenomenonNewfindingofRibosomebindingsiteorSD-sequence

Scientificresearch:Question:howcanthecellrecognizethecorrectstartcodon?AUGAUGAUGstartcodonordinarycodonAUGAUGAUGstartcodonordinarycodonSD-sequence2026/5/4411-3EukaryoticmRNAaremodifiedattheir5’and3’endstofacilitatetranslationpolypeptidemRNA戴帽穿鞋2026/5/442RNAprocessing：

5’endcappingThe“cap”:amethylatedguaninejoinedtotheRNAtranscriptbya5’-5’linkage

(甲基化的鸟嘌呤）2026/5/443OnceKozaksequenceEukaryoticmRNAusesamethylatedcaptorecruittheribosome.Oncebound,theribosomescansthemRNAina5’-3’directiontofindtheAUGstartcodon---scanning(扫描)2026/5/444Kozaksequence：5-G/ANNAUGG-3+1-3+4

HowwastheKozaksequencefound?

HowcanweprovethattheKozaksequenceisimportanttotranslation?

Question:HowwastheKozaksequencefound?

PhenomenonRaisequestionNewfindingofKozaksequence

AUGAUGAUGstartcodon5’capribosomescanningAUGAUGAUGstartcodon5’capribosomescanningKozaksequence：5-G/ANNAUGG-3startcodon+1-3+4Nextquestion:IstheKozaksequencereallyimportanttotranslationinitiation?

HowcanweprovethattheKozaksequenceisimportanttotranslation?

ExperimentalevidenceforprovingtheimportanceoftheKozaksequence:KozakM.PointmutationsdefineasequenceflankingtheAUGinitiatorcodonthatmodulatestranslationbyeukaryoticribosomes.Cell,1986,44:283-292研究论文（原始研究工作）的学习2026/5/449不仅仅知道分子生物学知识是什么？而且也了解分子生物学知识是怎么得来的？有哪些实验证据支持所学的基本理论和概念？不仅仅学习基本知识和概念，也学习科学研究的方法，接受科学思维的训练从别人的研究中获得启发，获得新的IDEA加深对基本概念的理解为什么要学习研究论文的研究工作？Ideaofmutation(geneticmethod)Kozaksequence：5-G/ANNAUGG-3startcodon+1-3+4MutatedtoothernucleotideDetecttheeffectofmutationontranslationefficiency研究思路：Designoftheexperiment5-G/ANNATGG+1-3+4Model：ratpreproinsulingene(小鼠前胰岛素原基因）ratpreproinsulingeneCOScellsgrowinginmediumcontaining[35S]-Methionine(甲硫氨酸）Detecttheamountofpreproinsulinprotein(35S

labeled)

IntroduceintoCOScell5-G/ANNATGG+1-3+4ratpreproinsulingenemutant+2+3+1protein

G/ANNATGG+1-3+4ratpreproinsulingene

G/ATTATGGstartcodonATGATGATGATGATGATGATGATG+1-3+4CTGprotein2026/5/454TheKozaksequenceisreallyimportanttotranslationinitiationConclusion:Poly-Ainthe3’endpromotestheefficientrecyclingofribosomes,thusincreasingthetranslationefficiency2026/5/456Basicconcepts:ORF:startcodon,stopcodon,monocistrionic,polycistrionicProkaryoticmRNA:ribosomebindingsite

(核糖体结合位点)EukaryoticmRNA:

amethylatedcaptorecruittheribosome,scanning,Kozaksequence

2026/5/457Topic2:tRNAThegeneticinformationinmRNAcannotberecognizedbyaminoacidsdirectly(thechaingroupofaminoacidcannotinteractwithbasegroupofmRNA).Thegeneticcodehastoberecognizedbyanadaptormolecule(transferRNA),andthisadaptorhastoaccuratelyrecruitthecorrespondingaminoacid.

2026/5/458tRNAfunctionNucleotidecodonaminoacidadaptor(转配器)tRNAThestoryoffindingtRNACrick’shypothesis:AdaptormolecularCanrecognizebothnucleotideandaminoacidAtypeofsmallRNAofunknownfunctionTheresearchworkofZamecnikin19572026/5/461TheresearchworkofZamecnikin1957Model:acell-freeproteinsynthesissystemfromratOneofthecomponentsofthissystemwasaso-calledpH5enzymefractionthatcandirectthemRNAtranslationMostofpH5enzymefractionwereproteins,but

ZamecnikdiscoveredthatthismixturealsoincludedasmallRNA

ThisRNAcouldbecoupledtoaminoacidMixthesmallRNAwiththepH5enzyme,ATPand[14C]leucineMorelabeledleucineaddedtothemixture,themorewasattachedtotheRNA

[14C]leucine[14C]RNAAperfectcorrespondencebetweenthelossofradioactiveleucinefromRNAandgainoftheleucinebythemicrosomeproteinIncorporationofleucinefromleucyle-tRNAintoproteinonribosome

Mixingthe[14C]leucine-chargedRNAwithmicrosome(containingribosome)14C放射性强度时间2026/5/466WhynatureselecttRNAastheadaptormolecule?

Watson-Crickprinciple:basepairing2026/5/467EachtRNAisattachedtoaspecificaminoacidsandeachrecognizesaparticularcodoninthemRNA2-1:tRNAareadaptorsbetweencodonsandaminoacids2026/5/4681.tRNAsare75-95ntinlength.

2.Therearemanymodifiedbases(修饰碱基),whichsometimesaccountingfor20%ofthetotalbasesinonetRNAmolecule.Primarystructure2026/5/469Fig14-3unusualbasesPseudouridine(

U，假尿嘧啶)isamodifiedbase.ThesemodifiedbasesintRNAleadtoimprovedtRNAfunction2026/5/470formfourstems(arms)andthreeloopsSelf-complementaryregionswithintRNA2-2:tRNAsshareacommonsecondarystructurethatresemblesacloverleaf(三叶草型)2026/5/471ThesiteofattachmentofaminoacidAthree-nucleotide-longsequenceresponsibleforrecognizingthecodonbybasepairingwithmRNAcloverleafstructureoftRNA2026/5/4722026/5/4732-3:tRNAshaveanL-shaped3-DstructureFig14-5the3-DstructureoftRNA2026/5/474Theanticodonloopatoneendandtheaminoacidacceptor

siteattheother.Thebasepairingisstrengthenedbybasestackinginteractions.2026/5/475Topic3:attachmentofaminoacidstotRNAAminoacidsshouldbeattachedtotRNAfirstbeforeaddingtopolypeptidechain.ChargedtRNA(负载tRNA)

UnchargedtRNA(空载tRNA)2026/5/4763-1tRNAsarechargedbyattachmentofanaminoacidtothe3’terminalAofthetRNAviaahighenergyacyllinkage(高能酰基)2026/5/477AminoacyltRNAsynthetase(氨酰tRNA合成酶)catalyzethisreaction:hasthreebindingsitesforATP,aminoacidandtRNA

2026/5/4783-2AminoacyltRNAsynthetaseschargetRNAintwostepsAdenylylation(腺苷酰化)ofaminoacids:transferofAMPtotheCOO-endoftheaminoacids.2.

tRNAcharging:transferoftheadenylylatedaminoacidstothe3’endoftRNA,generatingaminoacyl-tRNAs(氨酰tRNA).2026/5/479Step1-Adenylylationofaminoacids(腺苷酰化氨基酸）腺苷酰化氨基酸2026/5/480Step2-TransferoftheadenylatedaminoacidtotRNAaminoacyl-tRNAs(氨酰tRNA)tRNAGlu2026/5/4813-3：

eachaminoacyltRNAsynthetaseattachesasingleaminoacidstooneormoretRNAsEachofthe20aminoacidsisattachedtotheappropriatetRNA(s)byaminoacyl-tRNAsynthetases.Mostaminoacidsarespecifiedbymorethanonecodon,andbymorethanonetRNAaswell(isoacceptingtRNAs,同工tRNA).2026/5/482Thespecificitydeterminantsforaccuraterecognitionareclustersattwodistinctsites:theacceptorstemandtheanti-codonloop.2026/5/483Fig14-7鉴别碱基2026/5/4843-4Aminoacyl-tRNAformationisveryaccurate:selectionofthecorrectaminoacid(识别正确的氨基酸)

tRNAaminoacyltRNAsynthetaseaminoacidWhytheaminoacyltRNAsynthetasemustrecognizetheaminoacidandtRNAwithveryhighaccuracyandfidelity?Question:tRNAaminoacyltRNAsynthetaseaminoacid2026/5/4863-5RibosomesisunabletodiscriminatebetweencorrectlyorincorrectlychargedtRNAs(核糖体不能够识别是否携带正确的氨基酸)Theribosome“blindly”acceptsanychargedtRNAthatexhibitspropercodon-anticodoninteraction,whetherornotthetRNAischargedwiththecorrectaminoacid2026/5/487Oneclassicbiochemicalexperiment半胱氨酰tRNA丙氨酰tRNA半胱氨酸反密码子半胱氨酸反密码子2026/5/488mutation2026/5/489Thetranslationmachineryreliesonthehighfidelity(高保真）ofaminoacyltRNAsynthetasetoensuretheaccuratedecodingofmRNA2026/5/490Topic4:theribosomeTheribosomeisthemacromolecularmachinethatdirectsthesynthesisofproteinsTheribosomeiscomposedofatleastthreeRNAmolecularsandmorethan50differentproteins.2026/5/491翻译的基本装置之一：进行蛋白质翻译的的重要细胞器，合成蛋白质的车间2009年来自英国，美国和以色列的三位科学家正是因为“对核糖体的结构和功能的研究”而获得诺贝尔化学奖2026/5/498Couplingoftranscriptionandtranslationinprokaryotesribosome2026/5/4994-1theribosomeiscomposedofalargeandasmallsubunit2026/5/41002026/5/41012026/5/4102Thelargesubunitcontainsthepeptidyltransferasecenter(肽基转移酶中心),whichisresponsiblefortheformationofpeptidebonds.ThesmallsubunitinteractingwithmRNAcontainsthedecodingcenter(解码中心),

inwhichchargedtRNAsreador“decode”thecodonunitsofthemRNA.2026/5/41034-2:thelargeandthesmallsubunitsundergoneassociationanddissociationduringeachcycleoftranslation.2026/5/4104Ribosomecycles(核糖体循环):

ThesmallandlargeribosomesubunitsassociatewitheachotherandtranslatethemRNA,thendissociateaftereachroundoftranslation2026/5/4105OverviewoftheeventsofribosomecyclemRNA/initiatortRNA/smallribosomalsubunitpolypeptide2026/5/41064-3NewaminoacidsareattachedtotheC-terminusofthegrowingpolypeptidechain.ProteinissynthesizedinaN-toC-terminaldirection2026/5/41074-4

Peptidebonds(肽键)areformedbytransferofthegrowingpeptidechainfrompeptidyl-tRNA(肽酰tRNA)toaminoacyl-tRNA(氨酰tRNA)2026/5/4108NewpeptidebonePeptidyltransferasereaction(肽酰转移酶反应)N-terminalC-terminal2026/5/41092026/5/41104-5RibosomalRNAs(rRNA)arebothstructuralandcatalyticdeterminantsoftheribosomesrRNAismuc