《英漢對照分子生物學(xué)導(dǎo)論》課件 王勇 Chapter 5 mRNA Modifications in Eukaryotes、Chapter 6 Translation

Inprokaryotes,transcriptionproducesanearlyexactmRNAcopyoftheDNA,andthetranscriptisimmediatelytranslatedintoprotein.Ineukaryotes,aseriesofmodificationsoccurtomRNAduringandaftertranscription.在原核生物中，轉(zhuǎn)錄產(chǎn)生的mRNA幾乎是DNA的準(zhǔn)確拷貝，并且這一轉(zhuǎn)錄產(chǎn)物會立即被轉(zhuǎn)譯成蛋白質(zhì)。在真核生物中，轉(zhuǎn)錄時以及轉(zhuǎn)錄后會對mRNA進(jìn)行一系列修飾。Chapter5mRNAModificationsinEukaryotes

第5章真核生物mRNA的修飾5.1Capping5.2Polyadenylation5.3Splicing5.4mRNAEditing5.5Experiments5.1加帽5.2聚腺苷酸化5.3剪接5.4mRNA編輯5.5實驗研究Chapter5mRNAModificationsinEukaryotes

第5章真核生物mRNA的修飾5.1Capping/加帽AnmRNAthathasbeentranscribedbutisnotyetreadyfortranslationiscalledapre-mRNA,oraprimarytranscript.Cappingistheprocessofaddingaderivativeofguaninenucleotidetothe5’endofthepre-mRNA.一條已經(jīng)轉(zhuǎn)錄出來但還沒有準(zhǔn)備好用于轉(zhuǎn)譯的mRNA稱為前體mRNA或初級轉(zhuǎn)錄本。加帽是在前體mRNA的5’末端加上一個鳥嘌呤核苷酸衍生物的過程。Structureofthecap/帽的結(jié)構(gòu)5’-3’phospho-diesterbond5’-5’triphosphatebondRNApolymeraseIIDiscussedinChapter4Cappingtakesplacequiteearly.5’-CapRNApolymeraseIIRNADNACappingprocess/加帽過程CTD:C-terminaldomainRNApolymeraseIIDNAMethyltransferaseGuanylyltransferaseRNAtriphosphataseFunctionsofthecapstructure

帽結(jié)構(gòu)的功能1.Helpspreventdegradation

幫助防止降解2.Helpstransportintocytoplasm

幫助轉(zhuǎn)運到細(xì)胞質(zhì)中3.Enhancestranslation/增強轉(zhuǎn)譯4.Helpsremovethefirstintron

幫助去除第一個內(nèi)含子1.Helpspreventdegradation

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

幫助轉(zhuǎn)運到細(xì)胞質(zhì)中DiscussedinChapter73.Enhancestranslation/增強轉(zhuǎn)譯Cap-bindingproteinNotranslationoccurs.RNApolymeraseIIRNADNA4.Helpsremovethefirstintron

幫助去除第一個內(nèi)含子ThefirstintronAAAAAAA------AAAA5.2Polyadenylation

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

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

hasatypicaltypicalsequenceofAAUAAA.Thissequencegivesaninstructionas“tocutthemRNAabout20nucleotidesdownstream,nearaGU-richsequence”.

AAUAAAGUThecleavagecomplex/切割復(fù)合體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

基本的剪接反應(yīng)5’

AG/GUAUGU…bodyofintron…UACUAAC-YAG

/3’SplicesitesinyeastSplicesites:Sequencesthatmarkthebeginningandendsofintronsandexons.Thebasicsplicingreaction5.3.2ProteinsinvolvedinSplicing

在剪接中發(fā)揮作用的蛋白質(zhì)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嗜熱四膜蟲

SplicingthatoccurswithoutthehelpofproteinsorsnRNPsiscalledself-splicing.Self-splicingofgroupIintronSelf-splicingofgroupIIintron

II類內(nèi)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

內(nèi)含子存在的原因Introns-earlytheoryIntrons-latetheoryIntron:“WhyamIhere?”Intron-earlytheory/內(nèi)含子早現(xiàn)說E.coli“IheardsomebodysayE.colioncehadintrons.Isthattrue?”“Couldbe.Whoknows?!”Intron-latetheory/內(nèi)含子后現(xiàn)說E.coli“Wearetheoneshavingintrons!

Weareproudofit.”Alternativesplicing/可變剪接Alternativesplicing:akindofsplicingthatcanproducevariousproteinsfromonegene.DrosophilaDscamgeneExon3Exon54.14.24.34.44.54.64.74.84.94.104.114.12Exon4Exon6Exon9AlternativeSplicingGoesMainstream可變剪接成為主流TheScientist,Volume17(24):28,Dec.15,2003Alternativesplicing/可變剪接5.4mRNAEditing/mRNA編輯Sleepingsicknessisoneofthemostneglectedhumandiseases,andmainlyaffectsthemostdeprivedpeoplesofsomeAfricancountries.

Trypanosome

錐蟲RNAeditingintrypanosomeC

Uediting

/C

U編輯Intheliver…

/在肝臟中…Inthesmallintenstine…5.5Experiments/實驗研究HybridizationbetweenmRNAwithDNAmRNA與DNA之間的雜交DetectionofintronswithDNase

Hybridizationbetweenpre-mRNAandDNA

前體mRNA與DNA之間的雜交Chapter6Translation/翻譯Chapter6Translation/翻譯6.1TheGeneticCode6.2MechanismofTranslationinProkaryotes6.3TranslationinEukaryotes6.4tRNAStructureandWobble6.5Experiments6.1遺傳密碼6.2原核生物翻譯機理6.3真核生物翻譯6.4tRNA結(jié)構(gòu)與搖擺6.5實驗研究6.1TheGeneticCode /遺傳密碼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/遺傳密碼6.2MechanismofTranslation

inProkaryotes

原核生物翻譯機理6.2.1Initiation/

起始6.2.2Elongation/

延伸6.2.3Termination/終止Compositionofprokaryoticribosome

原核生物核糖體的組成6.2.1Initiation/起始IF-1DissociationIF-3NoreassociationSeparationof30Sand50SsubunitsSDsequence:thetranslationstartsignal16SrRNA3’endThe30sinitiationcomplex

30S起始復(fù)合體Thecollectionofthe30Ssubunit,themRNA,andtheinitiatortRNAiscalledthe30Sinitiationcomplex.TheinitiatortRNAThe70SinitiationcomplexThe70Sribosome6.2.2Elongation/延伸1)tRNAentering2)Peptidyltransfer3)Translocation4)tRNAleaving1)tRNAentering/tRNA進(jìn)入2)Peptidyltransfer/肽基轉(zhuǎn)移3)Translocation/移位4)tRNAleaving/tRNA離開Elongationfactors/延伸因子Weaver,MolecularBiology,Chapter18Elongationfactors/延伸因子Weaver,MolecularBiology,Chapter186.2.3Termination/終止Translationterminateswhenribosomereachesastopcodon,becausethereisnotRNAbeingabletobindtothestopcodon.Nowshowing

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

真核生物翻譯Compositionofeukaryoticribosome

真核生物核糖體的組成DifferencesinTranslationInitiation

翻譯起始的不同點InitiatortRNA/其始tRNAThescanningmodel/掃描模型1)InitiatortRNA/其始tRNACAUAAnticodonfMet-tRNAfMetFormylMetACC5’ACAGCA*

AlkylatedadenosineAnticodonMet-tRNAMet

MetACC5’AGCGCAG----引自：Turner,P.C.etal.《分子生物學(xué)精要速覽》（影印版），科學(xué)出版社，1999，p.249CAUProkaryoticinitiatortRNAEukaryoticinitiatortRNAProkaryoticinitiatortRNA

70SribosomeEukaryoticinitiatortRNA

80SribosomeCH3Yeast80SribosomeFile:Yeast80Sribosome1S1I.val2)Thescanningmodel/掃描模型Prokaryotes:SDsequenceEukaryotes:ScanningmodelThescanningmodel/掃描模型TranslationinitiatedbyenteringIRESsitedirectly通過直接進(jìn)入IRES位點啟動翻譯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