第14章蛋白质的生物合成与修饰.

1、第十四章妥匂质的生肠合成与修饰第一节概 述20世纪50年代的3个重要发现1 核糖体的发现：201比纪50年代初，Paul ZamecnikCrick o2.1RNA的发现:1957, Mahlon Hoagland and Zamecnik3.tRNA功能的确定: 20世纪50Nuelpntid triplet coiling fi)r an nmino acid年代末，Francis第二节遗传密码的破译三联体密码与阅读框(一)密码子(codon)为核昔酸三联体：20世纪60年代前，己知至少需要三个DNA 的核昔酸残基编码一个氨基酸。DNA的4种核昔酸(A, G, C, T)1 密码子为1个核

2、昔酸-> 仅编码4种氨基酸；2 .密码子为2联体-42= 16种组合；3 密码子为3联体->43=64种组合。（二）研究证明，密码子是不重叠（overlap）的, 密码子间无标点符号（punctuation ）:（三）阅读框（reading fnimc ）:连续的三联体密码 线性序列中，第一个密码子建立一种阅读框。二、人工合成多核昔酸和无细胞体系蛋白质合成 1961 年，Marshall Nirenberg 和 Heinrich Matthaei 利用多核甘酸磷酸化齣合成一条山相同核甘酸组 成的多核昔酸链，用它作模板，利用E.coli细胞 体系和GTP在体外合成蛋白质。发现poly

3、(U)编码 多聚Phe, poly(A)编码多聚Lys, poly (C)编码多 聚 Pro。Incorporation of Amino Acids into Polypeptides in Response to Random Polymers of RNA*Amino acidObserved frequency of incorporation (Lys = 100)Tentative assignment for nucleotide composition of corresponding codonExpoetod froqucncy of incorporation based

4、 on assignment (Lys = 100)Asparagine2420Glutamine24A?C20Histidine6ac24Lysine100AAA100Proline7ac2.ccc4.8ihreomne2ba2c. ac224Presented here is a summary of data Iron one of the early experinents designed to cuodotc tho genetic cede A sjntlwtic RNA containing only A and C residues in a 5： 1 fatio direc

5、ted polypeptide synthesis. Both tne identity and the quantity of amino acids ircorporMed were determined Based on the relative abundance of A and C residues m the synthetic RNA. and assigring the codon AAA (the most likely codcn) a frequercy cf 100, there should be three differ©n, codons of ccm

6、pcsition A?C each at a relative frequency of 20； three codons of composition ACZ. och a relative frequency of 4 0： arxl codon CCC at a roldtivo froqjoncy of O.fl. The CCC assigriment was based on mformotion derived from prior studs with poly(C). Where two tertative codor assignments are made, both a

7、re proposed to code for the same amine acidNote tnat these designations of nucleotide conpcsition conta n no formation on nudeotide sequence (except, of course. AAA and CCC)三、三核昔酸诱导氨酰IRNA对核糖体的特异结合 1964 年，Nirenberg 和 Philip Leder 发现，相应的已 知序列的三核苗酸存在时，游离的Em"核糖休会与 一个特异的氨基酰（RNA结合确定三联体密码。严怕27-2Trinuc

8、leotides Induce Specific Binding of Aminoacyl-tRNAs to Ribosomesl4C-Labeled aminoacyl-tRNA bound to ribosome*TrinucleotidePtetRNAP"Lys-tRNA*1Pro-tRNAPmuuu4.600AAA07.70CCC003.1Source； Modifiod from Nircnbc乍 M & Leder. P (1964) RNA codo Acords and protc n synthesis. Scteoce 145, 1399rEach num

9、nwtne tacw gmh the amcinr ot txund -C incro刁when theindicated trinueleotide 25 present, relative to a control in which re trinuclectide 忧旺 <*lded.四、具有特定重复顺序多聚核糖核昔酸模板的合成 H. Gobind Khorana制备已知的核I：酸重复丿亍；列，以 此作为模板，在体外合成蛋白质，发现可牛成几种 重复的多肽链need in Res lymers with 汜 and Four BasesAUCAUCAUCAUClie fie-2 li

10、e He 'AUCAUCAUCAUC-> _SerSerSerAUCAUCAUCAUC 二 His-His His Tilmiclcoudt wp«QU(IJUCLCPhc). (Ser. (Lcu>,<AAG|tIW，<C«u),(lUGhCLeuL(Cys>. MIKGCC心(Hmu (ThrS,(GUA>(W (ctMiin twmiiMto)*(LACK.Er加 CMI. <Leulvuc人01味 CS)m (His)R(GAUJ,CA$p (Metk (chair tennindteTitraruxZti"

11、;(LAUC),(Tir-Lfiu-Stf-lleb,<IUAC)(leu-Leu-ltr-iyr ln(GUMI«Oi mid lr»pn«ic*»rSUAG).Di and inpopW“fMrnycleotide Po4ypeptid« products遗传密码表：1966年确定Second letter of codonUCAGFirstletter ofcodon(5# end)VVVPJwucuSnrUAUTvrUGUCymUUCuccScrUACT>rUGCCysUUAl4?UUCASerUAA7 PUGAStnpUI

12、JGl>?UUCGS«»rl：AGSlopCCGTrpCUUI4»UecuProCAUHimCGUArgcueLeuCCCProCACIllsCGCArgCUALeuCCAProCAAQinCGAArpCUGLeuCCGlBroCAGGinCGGA nrAUU11cACUThrAAUAsnAGUSerAlJCIhACCTinAACAmiA(；CSprAHAlieACATlirAAAAGAArgAUGMetACGThrAAGLvbAGGArgGUUVaiGCUAlaGAUAapGGUGlvGUOVaiGCCAlaGACAsp(；GCGlyGUAVaiGCAAl

13、aGAAGiuGGAG(yGUGVaiGCGAlaGAGQluGGGGly第三节遗传密码的几个重要特性遗传密码的几个重要特性1. 连续性：读码必须从亍-3,方向连续阅读，密码子(codon )间无核背酸间隔。2. 起始密码子(initiation codon)和终止密码子(termination codon ):(1 )起始密码子：AUG(Met)(2) 终止密码子：UAA, UAG,UGAo为无意义密 码子(nonsense codon )。3简并性(degeneracy ): 除Met和Trp外， 多数氨基酸有26 个密码子。Degeneracy of the Genetic CodeAm

14、ino KidNumbr of codoniAla4他6Asn2Asp2Cys2Gin2Glu2Gly4His2IX3Lou S62Mel1Ph®2Pro4Sar6Thr41>P1叶244摆动性：密码子的头两个核昔酸起决定作用。5通用性(universal):(1) 人类、E.coli、烟草、两栖类和病毒都使用相 同的密码子。(2) 但线粒体和叶绿体中个别密码子有例外：1 )起始密码：AUG、AUU (Met)2) 终止密码：AGA、AGG3) Trp： UGAo二、翻译移码和RNA编辑Leu Gly Leu ArgLeu TlurAsn Leu Scopframe 5*u a

15、|<i g 0-|c v <J| c a < u r < 4 c a a it it r a u a g a h a g g g c c a 3沁 rtadiw frame cuagggcuccgcuugacaaauuuau ag ggaggccca I * Gly Am Ah劳氏肉瘤病毒的Clpol基内匝叠区2.RNA编辑原生动物EdTmRNAX IA A Al丽 T Al丽"A 什 A A (厂 TllQ T Al Ljrb Vai <Jiu A n Ia-h si四膜虫线粒体细胞色素氧化酚亚基II初始转录物的编辑(a) 四个U插入造成阅读框的改变(

16、b) 指导RNA£编辑产物互补，可能作为编辑模板脊椎动物HiimAn lizr 5*'、'UpdMOOiGm Leu QiuTLF矿Hum iii inlelineIf A A C V G C A-G Gin Lvii (Hu丄OH A VThr TyrRuxidiH iMlOibw2.1462.1442.150人低密度脂蛋AapoB-100基因转录物的RNA编辑: 小肠含有一种胞卩密唳脱氨爾，将C-U三、tRNA对密码子的识别1反密码子与密码 子的结合方向3iRNAmRNA 5' -A U C 】3,d 2釦Codon(a>AnticodonCodon

17、321(3 J G-C- I(5f) C-G-AG Tc321G-C-I (5JC-G-C (3)(b>2.1（肌苻酸）的存在3Cick的碱基摆动假说：(1)编码特异性：密码子的前两个碱基与反密码 子的相应碱塞j杉成强有力的Watson-Crick碱基肓止 对.(2) 反密码子的第一个碱基决定给定tRNA所读密码 子的数目.反密码子第一个碱基为C (A):配对专一；U (G):配对较不专一(阅读2个密码子) I：配对不专一(阅读3个密码子).(3) 当一个氨基酸由儿种密码子编码时，头两个碱 基不同的密码子需不同的tRNA.(4) 翻译61种密码子，最少需32种tRNA.严1 27-5Ho

18、w the Wobble Base of the Anticodon Determines the Number of Codons a tRNA Can Recognize*1. One codon recognized：Anticodon(3)X-Y-C (50(3。X-Y-A )Codon(5 ) Y-X 6(3f>Y-X-U (3J2 Two codons recognizcd： Anticodon(3')X-Y U(5J(3JX-Y G(5 mb MHCodon(5 ) YXY(3JY-X-g (3r)3 Three codons recognized： Anticod

19、on(3J X-Y-l )CocJon(5 ) Y-X- u )X and Y denote complementary bases capable ot strong Wa：son Cnck base painng with each other. The bases in the wobble positionsthe 3* position of codons and 5 position of rticcdorvs are shaded in rsd.四、遗传密码的天然改变Known Variant Codon Alignments in MitochondruCodons*UGAAU

20、AAGAAGGCUNCGGNormal code atugnmem蚀Ila盹Leu%An matsTrpStop叩Me:Se<+tYeastsIVpMet+Thr十TrpMe:+Thr?Sc匕gsacchbomyces pcvngTrp+4-+4-filarnontsus fungig+$IryDanocmesTrpH«h«< pnb-+Trp+?五、病毒DNA中不同阅读框的重叠基因为具有小基因组的牛物充分利用肓限遗传资源获 得最大化遗传信息的方式0X174噬茵体的重叠基因第四节蛋白质的生物合成蛋口质合成的五个阶段:氨基酸的激活c 女肽链的折叠与祓Compone

21、nts Required for the Five Major Stages of Protein Synthesis in E. coUStage1 Act Nation of 叭 g aci<%4 "Tcrminatior and5 Fofcjiog andprocowngEmentUI coman",20 ammo acitH20 ammoiK>-l tRNA20 Of fo俺 tRA%ATPMQmRNAM orr-ylrrftth oryMRNAInie.dKincodec in mRNA :AUC)305 rfoosoma $uUih«SOS

22、 rioosoma； subuntfInKmten t»(:tc<s IF-P iF-3)GTPMr*WxUd /OS ritwwme (inifintion cornpUA(r inodcv<specified 內 colonsE)cr Motion tactuis CEF-Tu. EF-Ts. EF-G)orn叱Termination cedcn in -1RKA，e*"x «!<*$ <Rf i,黄卜.RJATPSpecifc emzy<ne$ cogg 8 othe* 8mpoo初s for r«nofli ofli

23、Gstinc vendues andsequeicts addt>urio jxpfc«?»H4.Wation 伙 ternwooi roKlumeo >t1>ch(Dcnt of phoMto.carboxyl cxtobydrjte or prottt>et c grcxip%一、核糖体是一个复杂的超分子结构1.原核生物与真核 生物核糖体的比较16S rRXA mjcievl iw21 prntrtniilBSrHNA(1.900 nuckotidesi -33 |)rui»*iii»5SrRA120 nucklidoi 23

24、S rRNA1 idwl16 pmtrins2-M X l(rW.OJ) x lTM, 14 x I"BarU*riMl ribooaie 7BS V, 2»7 x l«FEukai*yotic rilM>«w>me os Mr x i(rOS rRNA <120mlcori w 288 rRNA<4.700 nudidYD :.NA< 160 ni*lco(H<*- -1H t- d»*m*2E.co16S 和 5SrRNA的二级结 构平面图（基于链 内碱基配对最大化 原则）3 核糖体上的结合位点 mRNA

25、结合位点 tRNA结合位点 P位点、A位点和E位点 GTP水解位点其他位点（多种蛋口合成因子和嗨的结合位点）2 结构特征：(1)二级结构: 三叶草型AntkodunCnntain* tun or thive D rpKi(iu« at diflerent positioivAulicodon armWobbleVaruibln in flizf% n«>t prnsnt in nil tRNAs二、tRNA具有特征性结构1简介：(1) 73-93个核昔酸(2) 相对分子量24 000-31 000(3) 一种氨基酸对应于一种以上的tRNAAC Anilno acidC

26、& Pu£(2) X-射线衍射测定的IRNA三维结构: 倒L型三、氨酰一tRNA合成酶和它们催化的反应1特点：氨酰7RNA合成酚对每个氨基酸及和应的 (RNA是专一的。2 催化反应式：3. 两类氨酰-IRNA合成酶27 8Two Classes of Aminoacyl-tRNA Synthetases*Class 1Class IIArgAlaCysAsnGinAspGluGlylieHisLeuLysMetPheTrpProTyrSerVaiThr-Here. Arg rep<esents arginyl-tRHA synthetase. arxJ so forth

27、 The clarification applies to all ocjBanisms for which tRNA synthetases ha/e been analyzed arxl is based on protein Uructural dKtifKti ons and on mcchanttic distinct Io n outlined in Figure 27-16.氨酰TRNA的-般结构1 校对功能的发现：用lie的相似物VM来检测Ile-tRNA合成酶对两 种底物的作用。结果显示，IQtRNA合成酚对lie 的结合优先于対Vai的结合2()()倍o门然中Vsl插入 l

28、ie位点的发生率是1/3000,这种梢确度就是出Ile tRNA合成嗨保证的。2 氨酰一 tRNA合成酶对tRNA氨酰化校对功能:Syrithetase $pp<?ilicfor isoleucmeValirc + AXPPPMistaken BGtioliovitRNA *ValineAMPE，i#Error co-rwcdunby hydrolycifiValire + AMP 十Figure 34 12Vi oofl eading ny hydrolv3i«s cf an in coi i ccl a !ui)uaGy；-A'MP'The enti v of RNA spccH ic for isoleuciMc induce hydrx>lyM5 ot valyl-AMP.五、氨酰-tRNA合成酶对tRNA的识别If第二遗传密码”:氨酰-tRNA合成酶与tRNA的和互作用。2.实验证