《英汉对照分子生物学导论》课件 王勇 Chapter 5 mRNA Modifications in Eukaryotes、Chapter 6 Translation

Inprokaryotes,transcriptionproducesanearlyexactmRNAcopyoftheDNA,andthetranscriptisimmediatelytranslatedintoprotein.Ineukaryotes,aseriesofmodificationsoccurtomRNAduringandaftertranscription.在原核生物中，转录产生的mRNA几乎是DNA的准确拷贝，并且这一转录产物会立即被转译成蛋白质。在真核生物中，转录时以及转录后会对mRNA进行一系列修饰。Chapter5mRNAModificationsinEukaryotes

第5章真核生物mRNA的修饰5.1Capping5.2Polyadenylation5.3Splicing5.4mRNAEditing5.5Experiments5.1加帽5.2聚腺苷酸化5.3剪接5.4mRNA编辑5.5实验研究Chapter5mRNAModificationsinEukaryotes

第5章真核生物mRNA的修饰5.1Capping/加帽AnmRNAthathasbeentranscribedbutisnotyetreadyfortranslationiscalledapre-mRNA,oraprimarytranscript.Cappingistheprocessofaddingaderivativeofguaninenucleotidetothe5’endofthepre-mRNA.一条已经转录出来但还没有准备好用于转译的mRNA称为前体mRNA或初级转录本。加帽是在前体mRNA的5’末端加上一个鸟嘌呤核苷酸衍生物的过程。Structureofthecap/帽的结构5’-3’phospho-diesterbond5’-5’triphosphatebondRNApolymeraseIIDiscussedinChapter4Cappingtakesplacequiteearly.5’-CapRNApolymeraseIIRNADNACappingprocess/加帽过程CTD:C-terminaldomainRNApolymeraseIIDNAMethyltransferaseGuanylyltransferaseRNAtriphosphataseFunctionsofthecapstructure

帽结构的功能1.Helpspreventdegradation

帮助防止降解2.Helpstransportintocytoplasm

帮助转运到细胞质中3.Enhancestranslation/增强转译4.Helpsremovethefirstintron

帮助去除第一个内含子1.Helpspreventdegradation

帮助防止降解RNaseRNase5’-5’triphosphatebond5’-3’phosphodiesterbond5’-3’phosphodiesterbond2.Helpstransportintocytoplasm

帮助转运到细胞质中DiscussedinChapter73.Enhancestranslation/增强转译Cap-bindingproteinNotranslationoccurs.RNApolymeraseIIRNADNA4.Helpsremovethefirstintron

帮助去除第一个内含子ThefirstintronAAAAAAA------AAAA5.2Polyadenylation

/聚腺苷酸化Poly(A)tailPolyadenylationPre-mRNAPolyadenylation

isamodificationprocessinwhichastringofabout250adeninenucleotidesisaddedtothe3’endofthetranscript.PolyadenylationdoesnotoccuratthenaturalendThepolyadenylationsignal/聚腺苷酸化信号AAAAAAA------AAAAPoly(A)tailPolyadenylationPre-mRNAPolyadenylationsignal

hasatypicaltypicalsequenceofAAUAAA.Thissequencegivesaninstructionas“tocutthemRNAabout20nucleotidesdownstream,nearaGU-richsequence”.

AAUAAAGUThecleavagecomplex/切割复合体CleavagecomplexCPSF(cleavageandpolyadenylationspecificityfactor)CstF(cleavagestimulationfactor)CFI(cleavagefactorI)CFII(cleavagefactorsII)Poly(A)-BindingProteinCTD:C-terminaldomainProteinsforcappingatCTDProteinsforpolyadenylationatCTDFunctionsofthepoly(A)tail

poly(A)尾的功能AAAAAAA------AAAAAAAAAAA---AAAAAAThemainfunctionofpoly(A)tailistoprotectthemRNAfromdegradationbyribonucleases.Functionsofthepoly(A)tail

poly(A)尾的功能AAAAAAA------AAAAThereisalsosomeevidencethatthepoly-Atailisinvolvedin

splicingandenhancestranslationofmRNAs.5.3Splicing/剪接Exons:Partsofagenethatareexpressedasprotein.Introns:Sequencesthatdonotcodeforproteinandinterruptthecodingregions.Splicing:Theprocessofremovingintronsfromapre-mRNA.5.3.1TheBasicSplicingReaction

基本的剪接反应5’

AG/GUAUGU…bodyofintron…UACUAAC-YAG

/3’SplicesitesinyeastSplicesites:Sequencesthatmarkthebeginningandendsofintronsandexons.Thebasicsplicingreaction5.3.2ProteinsinvolvedinSplicing

在剪接中发挥作用的蛋白质Spliceosome:Thecollectionoffactors,especiallysnRNPs,thathelpwiththesplicingofintrons.snRNPs:smallnuclear

ribonucleoproteinssnRNPsaresmallparticlesfoundinthenucleusandcontainbothproteinandRNA.snRNPsfunctioninginsplicing:U1,U2,U4,U5andU6.Nowshowing

mRNAsplicingFile:biophoto7\

LehningerPrinciplesofBiochemistry\mRNAsplicing5.3.3Self-Splicing/自我剪接Tetrahymenathermophilia嗜热四膜虫

SplicingthatoccurswithoutthehelpofproteinsorsnRNPsiscalledself-splicing.Self-splicingofgroupIintronSelf-splicingofgroupIIintron

II类内含子的自我剪接WithhelpofproteinsWithouthelpofproteinsSelf-splicingNormalsplicing5.3.4Trans-Splicing/反式剪接MaturemRNAProteinRNApieceofseparateoriginDNATrans-Splicing/反式剪接5.3.5ReasonsforIntrons

内含子存在的原因Introns-earlytheoryIntrons-latetheoryIntron:“WhyamIhere?”Intron-earlytheory/内含子早现说E.coli“IheardsomebodysayE.colioncehadintrons.Isthattrue?”“Couldbe.Whoknows?!”Intron-latetheory/内含子后现说E.coli“Wearetheoneshavingintrons!

Weareproudofit.”Alternativesplicing/可变剪接Alternativesplicing:akindofsplicingthatcanproducevariousproteinsfromonegene.DrosophilaDscamgeneExon3Exon54.14.24.34.44.54.64.74.84.94.104.114.12Exon4Exon6Exon9AlternativeSplicingGoesMainstream可变剪接成为主流TheScientist,Volume17(24):28,Dec.15,2003Alternativesplicing/可变剪接5.4mRNAEditing/mRNA编辑Sleepingsicknessisoneofthemostneglectedhumandiseases,andmainlyaffectsthemostdeprivedpeoplesofsomeAfricancountries.

Trypanosome

锥虫RNAeditingintrypanosomeC

Uediting

/C

U编辑Intheliver…

/在肝脏中…Inthesmallintenstine…5.5Experiments/实验研究HybridizationbetweenmRNAwithDNAmRNA与DNA之间的杂交DetectionofintronswithDNase

Hybridizationbetweenpre-mRNAandDNA

前体mRNA与DNA之间的杂交Chapter6Translation/翻译Chapter6Translation/翻译6.1TheGeneticCode6.2MechanismofTranslationinProkaryotes6.3TranslationinEukaryotes6.4tRNAStructureandWobble6.5Experiments6.1遗传密码6.2原核生物翻译机理6.3真核生物翻译6.4tRNA结构与摇摆6.5实验研究6.1TheGeneticCode /遗传密码Translation:synthesisofaproteinusinganmRNAasthetemplate.Transcription

tRNA:theconverter

tRNA:theconverter4basesvs.

20aminoacidsUCAGGlyAla

Ile

Leu

Met

Phe

Trp

Pro

Val

SerThr

Asn

Gln

Cys

Tyr

AspGlu

Arg

HisLys1base→1aminoacidUCAGGlyAla

Ile

Leu

Met

Phe

Trp

Pro

Val

SerThr

Asn

Gln

Cys

Tyr

AspGlu

Arg

HisLys2bases→1aminoacidUUUCUAUGGlyAla

Ile

Leu

Met

Phe

Trp

Pro

Val

SerThr

Asn

Gln

Cys

Tyr

AspGlu

Arg

HisLysCUCCCACGAUACAAAGGUGCGAGG3bases→1aminoacidThegeneticcode/遗传密码6.2MechanismofTranslation

inProkaryotes

原核生物翻译机理6.2.1Initiation/

起始6.2.2Elongation/

延伸6.2.3Termination/终止Compositionofprokaryoticribosome

原核生物核糖体的组成6.2.1Initiation/起始IF-1DissociationIF-3NoreassociationSeparationof30Sand50SsubunitsSDsequence:thetranslationstartsignal16SrRNA3’endThe30sinitiationcomplex

30S起始复合体Thecollectionofthe30Ssubunit,themRNA,andtheinitiatortRNAiscalledthe30Sinitiationcomplex.TheinitiatortRNAThe70SinitiationcomplexThe70Sribosome6.2.2Elongation/延伸1)tRNAentering2)Peptidyltransfer3)Translocation4)tRNAleaving1)tRNAentering/tRNA进入2)Peptidyltransfer/肽基转移3)Translocation/移位4)tRNAleaving/tRNA离开Elongationfactors/延伸因子Weaver,MolecularBiology,Chapter18Elongationfactors/延伸因子Weaver,MolecularBiology,Chapter186.2.3Termination/终止Translationterminateswhenribosomereachesastopcodon,becausethereisnotRNAbeingabletobindtothestopcodon.Nowshowing

Translation:thethreestepsFile:biophoto7\IntroductiontoGeneticAnalysis\Translation:thethreestepsoftranslation(5’)6.3TranslationinEukaryotes

真核生物翻译Compositionofeukaryoticribosome

真核生物核糖体的组成DifferencesinTranslationInitiation

翻译起始的不同点InitiatortRNA/其始tRNAThescanningmodel/扫描模型1)InitiatortRNA/其始tRNACAUAAnticodonfMet-tRNAfMetFormylMetACC5’ACAGCA*

AlkylatedadenosineAnticodonMet-tRNAMet

MetACC5’AGCGCAG----引自：Turner,P.C.etal.《分子生物学精要速览》（影印版），科学出版社，1999，p.249CAUProkaryoticinitiatortRNAEukaryoticinitiatortRNAProkaryoticinitiatortRNA

70SribosomeEukaryoticinitiatortRNA

80SribosomeCH3Yeast80SribosomeFile:Yeast80Sribosome1S1I.val2)Thescanningmodel/扫描模型Prokaryotes:SDsequenceEukaryotes:ScanningmodelThescanningmodel/扫描模型TranslationinitiatedbyenteringIRESsitedirectly通过直接进入IRES位点启动翻译IRES:internalribosomeentrysequencePicornavirus

/小核糖核酸病毒

www.uniklinikum-giessen.de/.../res_ires.htm

InternalentryAUGTranslationinitiationineukaryotes(1/4)eIFeukaryoticinitiationfactorTranslationinitiationineukaryotes(2/4)Translationinitiationineukaryotes(3/4)Translationinitiationineukaryotes(4/4)6.4tRNAStructureandWob