CHAPTER7Translation（第七章翻譯）12023/2/15TherevisedcentraldogmaTranslationRNAprocessingDNArepairandrecombination基因組的保持基因組的表達(dá)22023/2/15Whatistranslation?

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

protein:linearsequencesofaminoacidsinprotein

32023/2/15ThelanguageofmRNA:fouralphabet(A,G,C,U)Thelanguageofprotein:twentyalphabet(aminoacid)translation42023/2/15DNA：5-ATGAGTAACGCG-33-TACTCATTGCGC-5NontemplatestrandtemplatestrandtranscriptionmRNA：5-AUGAGUAACGCG-3translationProtein：Met-Ser-Asn-Ala52023/2/15TranslationextremelycostsInrapidgrowingbacterialcells,proteinsynthesisconsumes80%ofthecell’senergy50%ofthecell’sdryweightWhy?62023/2/15OutlineTopic1-4:Fourcomponentsoftranslationmachinery

mRNAtRNAattachmentofaminoacidstotRNA(aminoacyl-tRNAsynthetases)ribosomeTopic5-7:Translationprocess

initiation;elongation;terminationTopic8:Antibiotics(抗生素）andtranslation-嘌呤霉素72023/2/15BasicmachineryofTranslationmRNAs(~5%oftotalcellularRNA)-messageRNAtRNAs(~15%)-transferRNAaminoacyl-tRNAsynthetases(氨酰tRNA合成酶)ribosomes(核糖體):rRNA82023/2/15Whatisthekeyattributeofeachcomponent?Howthefourcomponentworktogethertoaccomplishtranslation?reductionism(還原論)holism(整體論)philosophy92023/2/15Questionsaddressedin

“BasicmachineryofTranslation”WhatistheorganizationofnucleotidesequenceinformationinmRNA?WhatisthestructureoftRNAs?HowdoaminoacyltRNAsynthetasesrecognizeandattachthecorrectaminoacidstoeachtRNA?Howdoestheribosomeorchestratethetranslationprocess?102023/2/15Topic1:mRNA(信使RNA)DNA3-nt-longcodons(三聯(lián)體密碼子)specifytheorderofaminoacidsmRNAtranscription112023/2/15DNA：5-ATGAGTAACGCG-33-TACTCATTGCGC-5NontemplatestrandtemplatestrandtranscriptionmRNA：5-AUGAGUAACGCG-3translationProtein：Met-Ser-Asn-Ala122023/2/151-1polypeptidechainsarespecifiedbyORF

openreadingframe(ORF,開放閱讀框):acontiguous,non-overlappingstringofcodonsEachORFspecifiesasingleproteinandbeginswithastartcodonandendswithastopcodon132023/2/15Thestartcodon（起始密碼子）

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

（閱讀框）forallsubsequentcodons142023/2/15startcodon152023/2/15Thestopcodon（終止密碼子）UAG,UGA,orUAA(5’-3’)Functions:1.DefinestheendofORF2.Signalterminationofpolypeptidesynthesis162023/2/15openreadingframe(ORF):

acontiguousstretchofcodons“read”inaparticularframe(decidedbystartcodon)

“open”totranslationbecauseitlacksastopcodon(untilthelastcodoninORF)172023/2/15EukaryoticmRNA(真核生物mRNA)monocistrionic,單順反子polypeptidemRNA182023/2/15ProkaryoticmRNA(原核生物mRNA)polycistrionic,多順反子Polypeptide-1Polypeptide-2Polypeptide-3Why?ORF1ORF2ORF3192023/2/15EukaryoticmRNA(monocistrionic,單順反子):onemRNAcontainsoneORFonemRNAProkaryoticmRNA(polycistrionic,多順反子):onemRNAcontainsmultipleORFsonemRNAOneploypeptideMultiplepolypeptides(relatedfunction)202023/2/151-2ProkaryoticmRNAshavearibosomebindingsite

(核糖體結(jié)合位點(diǎn))thatrecruitsthetranslationalmachinery(核糖體)原核生物mRNA結(jié)構(gòu)特點(diǎn)：212023/2/15Ribosomebindingsite(RBS)orSD-sequence:complementarywiththesequenceatthe3’endof16SrRNA.ORF1ORF2ORF3222023/2/15Ribosome(核糖體):rRNA232023/2/15Translationalcoupling(翻譯耦合)5’-AUGA-3’stopcodonstartcodonORF1ORF2ORF3overlapping24FindingofRibosomebindingsite(RBS)orSD-sequenceScientific

question:howcanthecellrecognizethecorrectstartcodon?AUGAUGAUGstartcodonordinarycodon2023/2/1525Model:R17phageinfectsE.coliR17phage:threegenes—codingforAprotein,coatproteinandreplicase(復(fù)制酶）Phenomenon:生物學(xué)現(xiàn)象E.coli

（大腸桿菌）ribosomecantranslateallthreephagegenesinvitro(體外）RibosomefromanotherbacteriumBacillus（芽孢桿菌）canonlytranslatetheAproteingeneWhy?2023/2/15262023/2/1527Ifthe30SsubunitisfromE.coli,phagecoatproteingenecanbetranslatedIfthe30SsubunitisfromBacillus,phagecoatproteingenecannotbetranslatedExperimentalresults:30Ssubunit（核糖體30S

小亞基）isthekeyfactordeterminingthetranslation2023/2/1528??2023/2/152930SsubunitwasfurtherdissociatedintoRNAandprotein

Ifthe16srRNAisfrom

E.coli,phagecoatproteingenecanbetranslatedIfthe16srRNAisfrom

Bacillus,phagecoatproteingenecannotbetranslated16srRNA

isthekeyfactordeterminingthetranslation2023/2/1530ORF1ORF2ORF3J.ShineandL.DalgarnofindtheSDsequence:2023/2/1531MorepowerfulexperimentalevidencefortheimportanceofSD-sequence

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

（人生長激素蛋白）2023/2/1532SD:GUGUGHumangrowthhormonegeneProducenohumangrowthhormoneproteinwildtypeE.colicell(16srRNA:CCUCC)突變的研究思想2023/2/1533SD:GUGUGHumangrowthhormonegeneProducemuchhumangrowthhormoneproteinmutatedtypeE.colicell

(16srRNA:CACAC)2023/2/1534Phenomenon（現(xiàn)象）Discoveryofmechanism（機(jī)制）behindthisphenomenonNewfindingofRibosomebindingsiteorSD-sequence

Scientificresearch:2023/2/1535Question:howcanthecellrecognizethecorrectstartcodon?AUGAUGAUGstartcodonordinarycodonAUGAUGAUGstartcodonordinarycodonSD-sequence2023/2/15362023/2/151-3EukaryoticmRNAaremodifiedattheir5’and3’endstofacilitatetranslationpolypeptidemRNA戴帽穿鞋372023/2/15RNAprocessing：

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

(甲基化的鳥嘌呤）382023/2/15OnceKozaksequenceEukaryoticmRNAusesamethylatedcaptorecruittheribosome.Oncebound,theribosomescansthemRNAina5’-3’directiontofindtheAUGstartcodon---scanning(掃描)392023/2/15Kozaksequence：5-G/ANNAUGG-3+1-3+4

HowwastheKozaksequencefound?

HowcanweprovethattheKozaksequenceisimportanttotranslation?

Question:40HowwastheKozaksequencefound?

PhenomenonRaisequestionNewfindingofKozaksequence

2023/2/1541AUGAUGAUGstartcodon5’capribosomescanningAUGAUGAUGstartcodon5’capribosomescanning2023/2/1542Kozaksequence：5-G/ANNAUGG-3startcodon+1-3+4Nextquestion:IstheKozaksequencereallyimportanttotranslationinitiation?

HowcanweprovethattheKozaksequenceisimportanttotranslation?

2023/2/1543ExperimentalevidenceforprovingtheimportanceoftheKozaksequence:KozakM.PointmutationsdefineasequenceflankingtheAUGinitiatorcodonthatmodulatestranslationbyeukaryoticribosomes.Cell,1986,44:283-292研究論文（原始研究工作）的學(xué)習(xí)2023/2/15442023/2/15不僅僅知道分子生物學(xué)知識是什么？而且也了解分子生物學(xué)知識是怎么得來的？有哪些實(shí)驗(yàn)證據(jù)支持所學(xué)的基本理論和概念？不僅僅學(xué)習(xí)基本知識和概念，也學(xué)習(xí)科學(xué)研究的方法，接受科學(xué)思維的訓(xùn)練從別人的研究中獲得啟發(fā)，獲得新的IDEA加深對基本概念的理解為什么要學(xué)習(xí)研究論文的研究工作？45Ideaofmutation(geneticmethod)Kozaksequence：5-G/ANNAUGG-3startcodon+1-3+4MutatedtoothernucleotideDetecttheeffectofmutationontranslationefficiency研究思路：2023/2/1546Designoftheexperiment5-G/ANNATGG+1-3+4Model：ratpreproinsulingene(小鼠前胰島素原基因）ratpreproinsulingeneCOScellsgrowinginmediumcontaining[35S]-Methionine(甲硫氨酸）Detecttheamountofpreproinsulinprotein(35S

labeled)

IntroduceintoCOScell2023/2/15475-G/ANNATGG+1-3+4ratpreproinsulingenemutant+2+3+1protein2023/2/1548

G/ANNATGG+1-3+4ratpreproinsulingene

G/ATTATGGstartcodonATGATGATGATGATGATGATGATG+1-3+4CTGprotein2023/2/15492023/2/15TheKozaksequenceisreallyimportanttotranslationinitiationConclusion:50Poly-Ainthe3’endpromotestheefficientrecyclingofribosomes,thusincreasingthetranslationefficiency2023/2/15512023/2/15Basicconcepts:ORF:startcodon,stopcodon,monocistrionic,polycistrionicProkaryoticmRNA:ribosomebindingsite

(核糖體結(jié)合位點(diǎn))EukaryoticmRNA:

amethylatedcaptorecruittheribosome,scanning,Kozaksequence

522023/2/15Topic2:tRNAThegeneticinformationinmRNAcannotberecognizedbyaminoacidsdirectly(thechaingroupofaminoacidcannotinteractwithbasegroupofmRNA).Thegeneticcodehastoberecognizedbyanadaptormolecule(transferRNA),andthisadaptorhastoaccuratelyrecruitthecorrespondingaminoacid.

532023/2/15tRNAfunctionNucleotidecodonaminoacidadaptor(轉(zhuǎn)配器)tRNA54ThestoryoffindingtRNACrick’shypothesis:AdaptormolecularCanrecognizebothnucleotideandaminoacidAtypeofsmallRNAofunknownfunction2023/2/1555TheresearchworkofZamecnikin19572023/2/15562023/2/1557TheresearchworkofZamecnikin1957Model:acell-freeproteinsynthesissystemfromratOneofthecomponentsofthissystemwasaso-calledpH5enzymefractionthatcandirectthemRNAtranslationMostofpH5enzymefractionwereproteins,but

ZamecnikdiscoveredthatthismixturealsoincludedasmallRNA

2023/2/1558ThisRNAcouldbecoupledtoaminoacidMixthesmallRNAwiththepH5enzyme,ATPand[14C]leucineMorelabeledleucineaddedtothemixture,themorewasattachedtotheRNA

[14C]leucine[14C]RNA2023/2/1559AperfectcorrespondencebetweenthelossofradioactiveleucinefromRNAandgainoftheleucinebythemicrosomeproteinIncorporationofleucinefromleucyle-tRNAintoproteinonribosome

Mixingthe[14C]leucine-chargedRNAwithmicrosome(containingribosome)14C放射性強(qiáng)度時(shí)間2023/2/15602023/2/15612023/2/15WhynatureselecttRNAastheadaptormolecule?

Watson-Crickprinciple:basepairing622023/2/15EachtRNAisattachedtoaspecificaminoacidsandeachrecognizesaparticularcodoninthemRNA2-1:tRNAareadaptorsbetweencodonsandaminoacids632023/2/151.tRNAsare75-95ntinlength.

2.Therearemanymodifiedbases(修飾堿基),whichsometimesaccountingfor20%ofthetotalbasesinonetRNAmolecule.Primarystructure642023/2/15Fig14-3unusualbasesPseudouridine(U，假尿嘧啶)isamodifiedbase.ThesemodifiedbasesintRNAleadtoimprovedtRNAfunction652023/2/15formfourstems(arms)andthreeloopsSelf-complementaryregionswithintRNA2-2:tRNAsshareacommonsecondarystructurethatresemblesacloverleaf(三葉草型)662023/2/15ThesiteofattachmentofaminoacidAthree-nucleotide-longsequenceresponsibleforrecognizingthecodonbybasepairingwithmRNAcloverleafstructureoftRNA672023/2/15682023/2/152-3:tRNAshaveanL-shaped3-DstructureFig14-5the3-DstructureoftRNA692023/2/15Theanticodonloopatoneendandtheaminoacidacceptor

siteattheother.Thebasepairingisstrengthenedbybasestackinginteractions.702023/2/15Topic3:attachmentofaminoacidstotRNAAminoacidsshouldbeattachedtotRNAfirstbeforeaddingtopolypeptidechain.ChargedtRNA(負(fù)載tRNA)

UnchargedtRNA(空載tRNA)712023/2/153-1tRNAsarechargedbyattachmentofanaminoacidtothe3’terminalAofthetRNAviaahighenergyacyllinkage(高能?；?722023/2/15AminoacyltRNAsynthetase(氨酰tRNA合成酶)catalyzethisreaction:hasthreebindingsitesforATP,aminoacidandtRNA

732023/2/153-2AminoacyltRNAsynthetaseschargetRNAintwostepsAdenylylation(腺苷?；?ofaminoacids:transferofAMPtotheCOO-endoftheaminoacids.2.

tRNAcharging:transferoftheadenylylatedaminoacidstothe3’endoftRNA,generatingaminoacyl-tRNAs(氨酰tRNA).742023/2/15Step1-Adenylylationofaminoacids(腺苷?；被幔┫佘挣；被?52023/2/15Step2-TransferoftheadenylatedaminoacidtotRNAaminoacyl-tRNAs(氨酰tRNA)tRNAGlu762023/2/153-3：

eachaminoacyltRNAsynthetaseattachesasingleaminoacidstooneormoretRNAsEachofthe20aminoacidsisattachedtotheappropriatetRNA(s)byaminoacyl-tRNAsynthetases.Mostaminoacidsarespecifiedbymorethanonecodon,andbymorethanonetRNAaswell(isoacceptingtRNAs,同工tRNA).772023/2/15Thespecificitydeterminantsforaccuraterecognitionareclustersattwodistinctsites:theacceptorstemandtheanti-codonloop.782023/2/15Fig14-7鑒別堿基792023/2/153-4Aminoacyl-tRNAformationisveryaccurate:selectionofthecorrectaminoacid(識別正確的氨基酸)

tRNAaminoacyltRNAsynthetaseaminoacid80WhytheaminoacyltRNAsynthetasemustrecognizetheaminoacidandtRNAwithveryhighaccuracyandfidelity?Question:tRNAaminoacyltRNAsynthetaseaminoacid2023/2/15812023/2/153-5RibosomesisunabletodiscriminatebetweencorrectlyorincorrectlychargedtRNAs(核糖體不能夠識別是否攜帶正確的氨基酸)Theribosome“blindly”acceptsanychargedtRNAthatexhibitspropercodon-anticodoninteraction,whetherornotthetRNAischargedwiththecorrectaminoacid822023/2/15Oneclassicbiochemicalexperiment半胱氨酰tRNA丙氨酰tRNA半胱氨酸反密碼子半胱氨酸反密碼子832023/2/15mutation842023/2/15Thetranslationmachineryreliesonthehighfidelity(高保真）ofaminoacyltRNAsynthetasetoensuretheaccuratedecodingofmRNA852023/2/15Topic4:theribosomeTheribosomeisthemacromolecularmachinethatdirectsthesynthesisofproteinsTheribosomeiscomposedofatleastthreeRNAmolecularsandmorethan50differentproteins.862023/2/15翻譯的基本裝置之一：進(jìn)行蛋白質(zhì)翻譯的重要細(xì)胞器，合成蛋白質(zhì)的車間2009年來自英國，美國和以色列的三位科學(xué)家正是因?yàn)椤皩颂求w的結(jié)構(gòu)和功能的研究”而獲得諾貝爾化學(xué)獎(jiǎng)872023/2/15882023/2/15892023/2/15902023/2/15912023/2/15922023/2/15932013年10月7日電瑞典卡羅琳醫(yī)學(xué)院在斯德哥爾摩宣布，將2013年諾貝爾生理學(xué)或醫(yī)學(xué)獎(jiǎng)授予美國科學(xué)家詹姆斯·羅思曼、蘭迪·謝克曼以及德國科學(xué)家托馬斯·祖德霍夫，以表彰他們發(fā)現(xiàn)細(xì)胞的囊泡運(yùn)輸調(diào)控機(jī)制。

諾貝爾獎(jiǎng)評選委員會在聲明中說，這三位科學(xué)家的研究成果解答了細(xì)胞如何組織其內(nèi)部最重要的運(yùn)輸系統(tǒng)之一——囊泡傳輸系統(tǒng)的奧秘。謝克曼發(fā)現(xiàn)了能控制細(xì)胞傳輸系統(tǒng)不同方面的三類基因，從基因?qū)用嫔蠟榱私饧?xì)胞中囊泡運(yùn)輸?shù)膰?yán)格管理機(jī)制提供了新線索；羅思曼20世紀(jì)90年代發(fā)現(xiàn)了一種蛋白質(zhì)復(fù)合物，可令囊泡基座與其目標(biāo)細(xì)胞膜融合；基于前兩位美國科學(xué)家的研究，祖德霍夫發(fā)現(xiàn)并解釋了囊泡如何在指令下精確地釋放出內(nèi)部物質(zhì)。2023/2/1594英國科學(xué)家約翰·奧基夫(JohnO’Keefe)、挪威科學(xué)家梅·布萊特·莫索爾(MayBrittMoser)和愛德華·莫索爾(EdvardMoser)獲得2014年諾貝爾生理學(xué)或醫(yī)學(xué)獎(jiǎng)。獲獎(jiǎng)原因：發(fā)現(xiàn)大腦中定位系統(tǒng)獲得諾獎(jiǎng)的三位科學(xué)家研究的是大腦中的“內(nèi)置GPS”——定位系統(tǒng)，它使人們可以在空間中進(jìn)行自我定位，也為更高級的認(rèn)知功能提供了細(xì)胞意義上的基礎(chǔ)。這三人的研究解答了“我們?nèi)绾蔚弥覀冊谀睦铩薄拔覀冊鯓诱业铰窂綇囊粋€(gè)地點(diǎn)到達(dá)另一個(gè)地點(diǎn)”以及“我們?nèi)绾蝺Υ孢@些信息，從而下次需要尋找相同路徑時(shí)可以立刻獲得它們”這幾個(gè)問題。諾獎(jiǎng)委員會在頒獎(jiǎng)聲明中說，這一發(fā)現(xiàn)解決了幾個(gè)世紀(jì)以來困擾著哲學(xué)家和科學(xué)家的問題：大腦究竟如何創(chuàng)造出周圍的空間地圖，而我們又如何在復(fù)雜的環(huán)境中進(jìn)行導(dǎo)向？諾獎(jiǎng)委員會表示，三位獲獎(jiǎng)?wù)哧P(guān)于大腦定位的研究成果有助于更好的理解某些疾病，譬如阿茨海默癥。阿茨海默癥的早期癥狀就是患者的方位意識開始減弱，而三位獲獎(jiǎng)?wù)叩难芯縿t能為這一疾病提供新的救治可能。2023/2/15952023/2/15Couplingoftranscriptionandtranslationinprokaryotesribosome962023/2/154-1theribosomeiscomposedofalargeandasmallsubunit972023/2/15982023/2/15992023/2/15Thelargesubunitcontainsthepeptidyltransferasecenter(肽基轉(zhuǎn)移酶中心),whichisresponsiblefortheformationofpeptidebonds.ThesmallsubunitinteractingwithmRNAcontainsthedecodingcenter(解碼中心),

inwhichchargedtRNAsreador“decode”thecodonunitsofthemRNA.1002023/2/154-2:thelargeandthesmallsubunitsundergoneassociationanddissociationduringeachcycleoftranslation.1012023/2/15Ribosomecycles(核糖體循環(huán)):

ThesmallandlargeribosomesubunitsassociatewitheachotherandtranslatethemRNA,thendissociateaftereachroundoftranslation1022023/2/15OverviewoftheeventsofribosomecyclemRNA/initiatortRNA/smallribosomalsubunitpolypeptide1032023/2/154-3NewaminoacidsareattachedtotheC-terminusofthegrowingpolypeptidechain.ProteinissynthesizedinaN-toC-terminaldirection1042023/2/154-4

Peptidebonds(肽鍵)areformedbytransferofthegrowingpeptidechainfrompeptidyl-tRNA(肽酰tRNA)toaminoacyl-tRNA(氨酰tRNA)1052023/2/15NewpeptidebonePeptidyltransferasereaction(肽酰轉(zhuǎn)移酶反應(yīng))N-terminalC-terminal1062023/2/151072023/2/154-5RibosomalRNAs(rRNA)arebothstructuralandcata