分子生物学第五章.ppt

第五章蛋白质翻译来源：不详5.1.基本元件5.2.Genetic Code5.3.peptide synthesize5.4.保证peptide准确翻译的机制5.5.Central Dogma 的发展5.1.基 本 概 念 the second step of gene expression Multiple&complex assessment tRNAas a adapter for codon&amino acid tRNAloading aa by aa-tRNAaa synthetase(AARS)&paracodon tRNArecognition codon by anti-codon codon;universal triplex codontwo of three reading codonparacodoncodon in codon codon degeneracy;wobble hypothesisisoacceptor codon usage(codon bias)mechanism of accurate translationinitiation,loading,elongation,proofreading5.2.基 本 元 件(Source:IrvingGeis/Peter Arnold,Inc.)5.1.1.tRNA mini RNA,4s,(70-80 Nt)tRNA phe,77Nt cloverleaf form(1964 HollyR.)5 arms&4 loops Nt more modified by methylation-DHUloop;contact withAARS-anti-codonloop;34th is wobble base-aaaccept arm;loadingaa at 3end-T Cloop;contactwith 5s rRNA-extraloop;classificationmarker?34I type;3-5 Nt 3/4 tRNAII type;13-21 Nt(来源：分子生物学（2007），郑用琏，第179页)CappingCap 0:m7GpppXpYpCap 1:m7GpppXmpYpCap 2:m7GpppXmpYmpHelp the splicing of the first intronPre-RNA tailingA poly(A)tail(50-200)be added at-20 Nt tailing signal(AAUAAA)from 3-end of Pre-RNASpecific endonulcease recognizes AAUAAA andthe following GUGUGUG,cuts within thesequence,adding poly(A)s at 3-endRNA internal methylation m5C m6A Modificationsof tRNAsPre-RNA splicing Introns be classified into I,II,III by junction sequence GroupI splicing model:5-exon-U-intron-G-exon-3CCS(centralcore sequence)Internalguidesequence(IGS):withinthe intron close to 5 junctionseq3 timesoftrans-esterificationbetweenG&URNA auto-splicingas Ribizyme GroupII splicing model:5-exon-GUGCG-B.S-Py AU-exon-3 GroupIII splicing model:5-exon-GU-intron-AG-exon-3,SnRNAs5-1tRNAmini RNA,4s,(70-80 Nt)Nt more modified by methylationtRNA phe,77Nt cloverleaf form Aa accept arm,DHU loop(contact with AARS),anti-codon loop,TC loop(contact with 5S rRNA),extra loop Paracodon:a numberof Nts,on tRNA,contact withAARSChapter 5 Protein translationParacodon-由若干Nt组成，存在于tRNA不定位置上-与AARS侧链基团的分子发生特异的“契合”-成为tRNA准确负载氨基酸的机制之一tRNA的”L”三维结构与功能“L”构型的结构力-二级结构中双链区的碱基堆积力和氢键-二级结构中非双链区在“L”结构中，形成氢键结合tRNA的”L”三维结构(来源：分子生物学（2007），郑用琏，第180页)“L”结构域的功能-aa accept arm 位于“L”的一端，契合于核糖体的肽基转移酶结合位点 P A,以利肽键的形成-anti-codon arm 位于”L”另一端，与结合在核糖体小亚基上的codonof mRNA配对aa-tRNAaa(来源：不详)-“L”结构中碱基堆积力大使其拓扑结构趋于稳定wobble base位于“L”结构末端堆积力小自由度大使碱基配对摇摆-T C loop&DHU loop位于“L”两臂的交界处，利于“L”结构的稳定Source:Quigley,G.J,Structural domains of transfer RNA moleculars,Science 194:197 have GC content of 60%&Rich methylation each cell contains from several hundred to over20,000 copies of rDNA gene rRNA synthesized in nucleolus and was stimulatedby low ionic strength&Mg+2Ribosomal genes(rDNA)are differentin several ways from other nuclear gene5.1.2.rRNAProkaryote 23s,16s,5s /Eukaryote 28s-5.8s,18s,5sRich methylation (m2U,m3A,m3U,m26A(二甲基)(来源：不详)5s RNA与T C loop of tRNA部分互补，并可配对In Prok.3-end of 16s rRNArich CCU plementary with5leading seq.of mRNAShine-Dilgarnoseq.of richAGG(来源：分子生物学（2007），郑用琏，第183页)23s rRNA-6 domains-有的与对抗生素的抗性有关-2660Ntregion -I loop(alpha Sarcin)binding with complex of aa-tRNAaa(EF)-TuGTP(引起核糖体变构！)G2661 C,aa-tRNAaa intoAsite go down-G2252,G2253双突变为C,将对转肽酶的活性产生抑制In Euk.3-end of 18s rRNA 与原核生物高度相似,但无与 S.D.seq.互补的保守序列在 mRNA的AUG上游存在CCACC核糖体scanningseq成为核糖体识别第一个AUG的信号AMEAMECCUGCGGUUGGAUGACCUCCUUBacterial16S18MammalianAMEAMECCUGCGGAAGGAUGAUUA高度相似5-end;300Nt leading seq.(A/G-AUG)richA,U,poly-cistronShine-Dalgarnoseq.(S.D seq)GGAGGS.D seq-AUG79Nt better G mut.translation go down5.1.3.mRNA In Prokaryote(Source:Molecular Biology(2002),Robert F.Weaver,Page539）In Eukaryotemono-cistronleading seq.5m7Gppp-CCACC-A-3-A1U2G3G4核糖体小亚基扫描AUG至关准确翻译的信号序列But mRNA of chloroplastshows similarities to prokaryotetype1;S-D seq.with greatersecondary structurein L.S.type2;richAU with littlesecondary structurein L.S.polycistron5.2.Genetic CodeSource:Oscar Miller/SPL/Photo Researchers,Inc5.2.1.Universal triplet codoncodon的特征codon是mRNA上连续排列的三个核苷酸序列，并编码一个氨基酸信息 的遗传单位codon具有四大生物系统的通用性与保守性（除mt）在一个基因序列中 codon具有不重叠性和无标点性In vitro Poly(U)Poly(C)Poly(A)Poly(G)Butpoly(UCUCUC)UCU/CUCpoly(Phe)peptidepoly(Pro)peptidepoly(Lys)peptidepoly(Gly)peptidepoly(Ser-Leu-Ser-Leu)Ser/Leu?密码子的破译 (1968.nobel prize)Marshall Nirenberg(1961)M.Nirenberg&P.Leder(1964.Science 145;1399)In vitroUCU(trinucleotides)Ser-C14,Leu,Lys,Arg,Ser,Leu-C14,Lys,Arg,Ser,Leu,Lys-C14,Arg,tRNAaaRibosomeNitrocellulose filterSer-C14.Leu-C14.Lys-C14.Gly-C14.(来源：分子生物学（2007），郑用琏，第188页)Stop codon 的证实61个codons被破译，(仅剩UAA,UAG,UGA？)Brenner(1961)获得；T4phage 头部蛋白基因的琥珀突变（amber）证明；突变体头部蛋白较野生型的变短推测；头部蛋白基因发生了终止突变，使蛋白质合成中断。Garen(1965)获得；E.coli 碱性磷酸酯酶基因（phoA）Amber突变株的大量回复突变株分析；回复突变株中对应“回复”的氨基酸发生终止突变的原氨基酸Trp(UGG)UACGln:CAG,CAAGlu:GAG,GAA证明：终止突变密码为UAG(amber 琥珀突变)UAA(ocher 赭色突变)UGA(opal 蛋白石突变)?!Stop codon 的证实aa and codon in back mutantSer:UCG,UCC,UCA,UCU,AGU,AGCLeu:UUG,UUA,CUU,CUC,CUA,CUGUAGUAGTyr:UAU,UAGLys:AAG,AAAUAGUAGUAG(来源：分子生物学（2007），郑用琏，第190页)5.2.2.Degeneracy of codon(密码子的简并现象)a)简并现象的概念；-一种氨基酸受2个以上codon编码的遗传现象-编码一种aa的4个codon间,仅3rd Nt 不同,称为 codon family例；Ser(6 codons)1 codon family&2 extra codons53 A Ab)简并现象的机理；Isoacceptor;负载同一氨基酸，但识别不同密码子的不同tRNAWobble hypothesis;GCGGCUUCU5353 AtRNA3 isoacceptors1 codon family 2 extra codons反密码子:密码子 在一定范围内的可选择配对现象1th(Nt34):3 rd-NtmRNA5-CGU-CGC-CGA-CGG-AGA-AGG-3wobble?!简并现象的机理；Isoacceptor;负载同一氨基酸，但识别不同密码子的不同tRNA负载同一氨基酸，识别相同密码子的不同tRNA？！Tyr codon:UACAUGAUG识别UAC Codon负载Try的tRNA有两个，但结构向差较大。YYtRNAmeti&tRNAmete ;tRNAmetf&tRNAmetm存在明显的结构差异553No base pairingtRNAmetftRNAmetmM3MG-C richG-C rich(来源：分子生物学（2007），郑用琏，第196页)CGCGCGCGU13(来源：分子生物学（2007），郑用琏，第193页)Wobble base的摇摆配对原则GUG（val）的第一Nt会以较低频率与tRNAmetf反密码子(CAU)发生“摇摆”配对，而作为起始密码.(E.coli GUG/AUG=1/30)(Source:Molecular Biology(2002),Robert F.Weaver,Page570）mRNA(1GUG)（val）作为起始密码.与tRNAfmet的反密码子(CA3U)配对，不是真正意义上的”摇摆”.由于tRNAmet f中反密码子下游第一个Nt(37)为未修饰的A，而其他tRNA第37个Nt几乎为较大的烷化修饰的Nt例如tRNAmet m第37个Nt为t6A(N6-苏氨酸羰酸腺苷)tRNAmeti&tRNAmete ;tRNAmetf&tRNAmetm存在明显的结构差异553No base pairingtRNAmetftRNAmetmM3MG-C richG-C richN6-苏氨酸羰酸腺苷(来源：分子生物学（2007），郑用琏，第196页)意味着反密码子边序碱基修饰对限制错读的机制AUGCA3UA（37）mRNAtRNAmetftRNAmetm1GUGCA3UA（37）AUGCAUt6A（37）碱基摇摆配对的方式C GU GThio-U GC IU AThio-U AU IssA I(Source:Molecular Biology(2002),Robert F.Weaver,Page570）Wobble base摇摆配对的机理-tRNA的拓扑空间结构34th摇摆位点位于拓扑结构的末端，碱基堆积力小，选择性配对的自由度大-34th摇摆位点被修饰的频率高导致配对原则的改变尤以A34 II=A/I=C/I=U-34th几乎无A-线粒体中U34=N(any)when U34 U*=A/G onlySource:Quigley,G.J,Structural domains of transfer RNA moleculars,Science 194:197Xo5UCmnm5UmCm5UXm5s2UK2C(5-羟基尿苷)(5-羧甲基氨甲基尿苷)(5-甲氧基羰甲基尿苷)(5-甲基-2硫代尿苷)(2-赖氨酸胞苷)Com5U(5(2)-羟羧甲基尿苷)I(Inosine次黄嘌呤)m7Gm5Cm6As2C(7-甲基尿苷)(5-甲基胞苷)(6-甲基腺苷)(2-硫代胞苷)(假尿苷)t6A (N6-苏氨酸羰酸腺苷)Q(Queuosine辫苷)5.2.3.Anti-codon及其两侧碱基修饰对密码子解读的生物学意义a)Methylated Nt at anti-codon and flankedb)被修饰的Nt34的配对能力Nt1 of anti-codon Nt3of codonU(mt,ct)CmO5U (5(2)-羟羧甲基尿苷)Cmnm5U(5-羧甲基氨甲基尿苷)mCm5U(5-甲氧基羰甲基尿苷)Um(2-O-甲基尿苷)Xm5S2U (5-甲基-2硫代尿苷)Q (Queuosine)I(Inosine）A,U,C,GA,G,UA,GA,GA,GAU,CU,C,Ac)tRNA中anti-codon碱基修饰的意义限制对密码识读的随意性，以保证遗传的稳定U A/GCm5S2U A(only)在特定表达细胞中S2U A提高摇摆能力，防止突变效应，以保证遗传的稳定A UU A/GAUI A/C/UCmO5U A/G/U5.2.4.tRNAabundance&codon usage(codon bias)Codon usage Observed for E.coli Ribosome Protein1209 codons(来源：分子生物学（2007），郑用琏，第197页)Codon usage in the genes of Animals2244codons(来源：分子生物学（2007），郑用琏，第198页)tRNA abundance&codon usage(codon bias)生物GC%不等各种codon的频率不等进化过程中度重复基因tRNA的拷贝数与codon使用频率的对应-识别同一氨基酸的不同tRNA(isoacceptor)量不等-不同生物间同一isoacceptor的量不等tRNAabundance;codon usage(codon bias)是进化中形成的基因表达调控机制之一tRNA abundance 正相关 codon usage)需要量多的蛋白质（除mRNA转录速率高外）有关aa的codonusage 高 相应tRNA量多需要量少的蛋白质（除mRNA转录速率低外）关键aa的codonusage 低相应tRNA量少）codon 与anti-codon间的作用强度codon usagemodulator ）需较长时间以求结合稳定intoAsite of ribosome融解温度高需较长时间Out Psite of ribosome自然选择codon/anti-codon间适度结合强度的codonusage以保证最佳的蛋白质合成速率G C 强氢键配对aa-tRNAaaG U 弱氢键配对aa-tRNAaathe seq.of codon in usage1 2-31 2-3in generalUUGAA CGG UCC A23/1209 10/120913/1209 3/1209Anti-codonAAG1AUG1In prok.Gly(GGG)usage=0Phe(UUC)(UUU)Pro(CCC)usage=0Tyr(UAC)(UAU)共性:codon/anti-codon间适度结合强度个性:G/C含量不同,tRNA丰度各异5.2.5.two of three codon-reading in mitochondriala)线粒体中具有与通用密码不同的编码信息线粒体codon较为整齐（均为2/4/6）2 codon;F,I,Y,H,Q,N,E,k,D,W,M,C4 codon;V,P,T,A,R,G,(family)&stop codon6 codon;L,S(2 isoacceptorseach)In mt 22 tRNA only(32 tRNA in universal code)线粒体“三中读二”方式可减少tRNAArgstopstopTrpIleMet(来源：不详)(来源：不详)Codon-readingForcodon family;two of three readingcodonanti-codonUCU-UCA-UCG-UCCUAGUC Ser codonN34(U)A/U/C/G仅起将codon隔开的作用stopTrpAGIleMetACGACGArgstopCCGGGUGC(来源：不详)Codon-readingFor 2 codon type;Nt34 wobble base G C/UArgGAAGAUIleMetGUUstopTrpGUGGUAGCAGCUstopGUC(来源：不详)Codon-readingFor 2 codon type;Nt34 wobble base*U G/AArgstopUAAIleMetUAUUUGUUUUUCstopTrpUCA(来源：不详)Codon-reading in mt(Nt34：U/U*/G)Forcodon family;two of three readingUC Ser codon,N34(U)仅起将codon隔开的作用For2 codon type;Nt34 wobble base U*G/AG C/Ucodonanti-codonUCU-UCA-UCG-UCCUAG一种GGC编码几种氨基酸蛋白质性质不变5.2.6.codon in codon or general genetic codon(GGC 广义密码子)生物体除具有标准的通用密码保证蛋白质的准确翻译外同时存在GGC转录的模糊性（非转录错误）生物的适应性a)codon/anti-codon间的缔合能分析2ed Nt of codon（N1N2N3）对codon/anti-codon的缔合能贡献最大凡2edNt相同的codoncodon/anti-codon间的缔合能相似对缔合能的贡献2Nt 1thNt 3rdNtb)codon对氨基酸性质的决定2edNt of codon 对氨基酸性质和蛋白质空间结构的决定度较大NUN 非极性疏水性氨基酸-helix&-sheet的形成者位于蛋白质分子内部NAN 极性亲水性氨基酸,位于蛋白质分子外部N（G/C）N 编码的氨基酸极性居中疏疏GGNCUNAUA/CGUNGCUUUU/CUGU/GAUGUGU/CUUU/CAUA/CGUNCUNAUGUGGCAU/CGGN MW 75 kd小GCNUCNCCNGUNCANUGU/CCUNACN中UAG/C中AUA/C中Codon in codon(依 2edNt of codon预测氨基酸的性质)不同方法测定aa的亲水性和分子量结果(1)N1N 2N 3(2)N1N 2N 3F.J.R.Taylor 1989 Bio-Systems 22.p177-187N1N 2N 3UCN中GCN中GAU/C中UGGUAU/CCAA/GAAA/GAAU/CGAA/GCAU/GGGNACNUCNCCNCGNAAU/CCAA/GAAU/CGAA/GCAA/GAAA/GAUGCAU/CUUU/C亲GAU/CCGNGAA/GGAU/C 亲AAA/GCGNUAU/CUGG大 204 kd2ed Nt =UA(hydrophobic aa)(hydrophilicaa)G/C (neutral aa)c)Nt of codon 对蛋白质功能的决定 1th&3rd Nt的摇摆对蛋白质的结构与功能影响不大 2edNt不能摇摆2edNt of codon对氨基酸的编码特征即为GGCor codon in codon蛋白质定点诱变蛋白质改造（蛋白质工程）d)生物学意义 保证遗传的稳定 依据codonin codon（2edNt of codon）判断蛋白质的性质 蛋白质性质预测(Source:TriposAssociates/Peter Arnold,Inc.)5.3.1.direction of peptide elongation N CR1 O H R2R3NCCNCC+NCCHHOOHHHOOHHHHR3R1 O H R2+HHOOHHHOOHNCCHNCCNCCHH机制？P O3Met+tRNAmetfACCA-O2 C OP OH3H C N HH CH HSCH3ACCA-OH2OH 2OH 3aa转酯CH3formylationC OH C N HH CH H OHSCfMettRNA metf&Met-tRNA metm5.3.2.AminoacyltRNAaain Prok.in Euk.MettRNA metI&Met-tRNA meteAARSAminoacyltRNAsynthetaseR非特异结合位点 DHU loop特异结合位点 paracodan(来源：分子生物学（2007），郑用琏，第211页)5.3.3.peptide synthesisIF-1IF-2IF-39.5kd95kd-117kd20kd加强IF-2，IF-3的酶活促使fMet-tRNA fmet 选择性的结合在30S亚基上促使30S亚基结合于mRNA起始部位(识别tRNA fmet 中富含GC的反密码子臂,way in Psite?!)具有解离30S与50S亚基的活性IF2fmetIF-2Binary complexfmetComplete30s-mRNA complexIF3GTPfmetInitiation complexIF3fmet(来源：不详)eIF2eIF2-AeIF1eIF3eIF4beIF4aeIF4CeIF5eIF4e3种亚基65kd15kd500kd80kd50kd19kd150kd(eIF4f 的亚基)形成三元起始复合体（eIF2,GTP,tRNA)促使Met-tRNAmet与40S亚基结合i促使mRNA与40S亚基结合促使mRNA与40S亚基结合促使mRNA与40S亚基结合促使与mRNA,GTP结合促使两亚基结合释放eIF2,eIF3与5端帽子结合(a):translation ofCapped Sindbis virus mRNA(b):translation ofUncapped picona virus mRNA(a)with eIF4ewithouteIF4e(C)(b)(d)eIF4e stimulates translation ofcapped,but not uncapped,(Source:Shatkin,Differential stimulation ofcapped mRNA translation in vitro by cap-binding protein Nature 285:331,1980.including 8 activation sites&occupy 20 Nt Psite(peptidyl attachment site)Asite(Aminoacyl binding site)E site(Exit site of tRNA)5s rRNAsite(5s rRNA+T C loop)转位因子EF/G binding site mRNA biding site peptididyl transferasebinding site 延伸因子复合体EF-Tu-aa-tRNAaa binding siteTranslation domainmenbraneExit domainExit5ssitesite AsitePeptidyl transferasefMet-tRNAmet way in Psitesite by S.D Seq.(prok.)EF-GsiteScanning sequence way in Psite.A,site20 Nt(来源：分子生物学（2007），郑用琏，第185页)fMetMetMet-tRNAmetiS.D.Sequence?Scanningsequence?CP AfMet-tRNAmetf(来源：不详)CCPAMetAPfMet(来源：分子生物学（2007），郑用琏，第205页)AntibioticKilles bacteria and other cellsfMet-tRNAMet occupiesPsite orAsite？(Source:Molecular Biology(2002),Robert F.Weaver,Page575）Chapter 5 Protein translationtRNA mini RNA,4s,(70-80 Nt)Nt more modified by methylation tRNA phe,77Nt cloverleaf formAa accept arm,DHU loop(contact with AARS),anti-codon loop,TCloop(contact with 5S rRNA),extra loopParacodon:a numberof Nts,on tRNA,contact with AARSrRNA High GC-content,rich methylation,high copy number,synthesized innucleolus Pro:23S+5S,16S;Euro:28S/5.8S+5S,18S5-1mRNA Pro:Shine-Dalgarnoseq.(S.D seq)GGAGG Euro:5 m7Gppp-CCACC-A-3-A1U2G3G4 Degeneracyof codon Codon family Mechanismof codon degeneracy Isoacceptor:different tRNA that load the same aa,but recognizedifferent/same codon Wobble hypothesis:34th Nt in tRNACodon usuage/biasmRNA Pro:Shine-Dalgarnoseq.(S.D seq)GGAGG Euro:5 m7Gppp-CCACC-A-3-A1U2G3G4 Degeneracyof codon Mechanismof codon degeneracy Isoacceptor:different tRNA that load the same aa,but recognizedifferent/same codon Wobble hypothesis:34th Nt in tRNACodon usuage/bias Differentcondonsare used at differentfrequencyby aspeciesPeptide synthesis Direction of peptide elongation AminoacyltRNAaa,Initiation and elongation AARSthree sites:tRNA site,AA site,ATPDHU loop,nonspecific;paracodon,specific Enzymes forIF1:separate 50S and 30S subunits,help other factorsIF2:for the bindingof fMet-tRNA fmet to 30SIF3:for the bindingof mRNA to 30S5-5Peptide synthesis Enzymes foreIF4e,cap bindingfactoreIF4e stimulates translation of capped,but not uncappedmRNAMet-tRNAMet occupies ribosomal P site&InitiationtranslationTu and TsPuromycin Resembles an aminoacyl-tRNA Can bind to the A site Couple with the peptide in the P site Release it as peptidyl puromycinIf peptidyl-tRNA is in the A site,puromycin will notbind to ribosome,peptide will not be releasedTwo sites are defined on the ribosome:Puromycin-reactivesite(P)Puromycinunreactive site(A)3rd site(E)for deacylated tRNA bind to E site as exitsribosomeAntibioticKilles bacteria and other cellsfMet-tRNAMet occupiesPsite orAsite？Mixed 35SfMet-tRNAfMet withribosomes,AUG,and puromycin(嘌呤酶素).If AUG attractedfMet-tRNAMet tothe P site,then the labeledfMetshould have been able to react withpuromycin(inAsite),releasing labeledfMet-puromycin.If the fMet-tRNAMet went to the A site,puromycin should not have been ableto bind,so no release of labeled aminoacid should have occurred.fMet-tRNAMet occupies ribosomalPsite&Initiation translation(Source:Bretscher and Marcker Nature 211:382-3,1966)Way in PWay inAunderelongationWay in PSecondary structure near the 5-end of anmRNA can have either positive or negativeeffectsHairpin just past an AUG can force a pause byribosomal subunit and stimulate translationVery stable stem loop between cap and initiationsite can block scanning and inhibit translationGiven the amount of control at the transcriptionaland posttranscriptional levels,why control geneexpression at translational level?Major advantage=speed New gene products can be produced quickly Simply turn on translation of preexisting mRNAValuable in eukaryotesTranscripts are relatively longTake correspondingly long time to make Most control of translation happens at theinitiation stepMost bacterial gene expression is controlled attranscription levelMajority of bacterial mRNA has a very shortlifetime Only 1 to 3 minutes Allows bacteria to respond quickly tochanging circumstancesDifferent cistrons on a polycistronic transcriptcan be translated better than othersmRNA secondary structure can governtranslat