《生物化学》教学课件：蛋白质的生物合成（翻译）

1、蛋白质的生物合成（翻译）Translation,第 十 八 章,上两章我们学习了DNA 和 RNA 的合成. 我们这章主要学习protein 合成的机制, 也叫蛋白质的翻译。,Translation的过程或概念要更复杂一些。 DNA and RNA synthesis 遵循碱基配对的原则。,蛋白质的合成严格依赖于核酸的序列和蛋白质因子。蛋白质的合成是在核糖体内完成的。核糖体是一个巨大的复合体，含有三种大的RNA分子和50多种蛋白质。,这些研究结果表明核糖体是一个RIBOZYME, 也就是说，RNA对核糖体功能起很重要的作用。,近几年生物化学的重大贡献是对核糖体的结构的揭示以及在原子的水平研究核

2、糖体功能。,Paul Zamecnik,aminoacyl tRNA synthetases,Mahlon Hoagland,1954年，Paul Zamecnik及其同事实验证明体内蛋白质是由氨基酸合成的。,主要发现：,发现：,1958年，M. B. Hoagland和Zamecnik又发现蛋白质生物合成需要可溶性RNA为中介。,transfer RNA (tRNA) Incidentally, that labs discovery of tRNA supported the theory of complementarity (molecular biology),发现：,After t

3、he structure of DNA was discovered by James Watson and Francis Crick, who used the experimental evidence of Maurice Wilkins and Rosalind Franklin (among others), serious efforts to understand the nature of the encoding of proteins began. George Gamow postulated that a three-letter code must be emplo

4、yed to encode the 20 standard amino acids used by living cells to encode proteins, because 3 is the smallest integern such that 4n is at least 20.,The fact that codons consist of three DNA bases was first demonstrated in the Crick, Brenner et al. experiment. The first elucidation of a codon was done

5、 by Marshall Nirenberg and Heinrich J. Matthaei in 1961 at the National Institutes of Health. They used a cell-free system to translate a poly-uracil RNA sequence (i.e., UUUUU.) and discovered that the polypeptide that they had synthesized consisted of only the amino acid phenylalanine. Nirenberg we

6、re able to determine the sequences of 54 out of 64 codons in their experiments.,They thereby deduced that the codon UUU specified the amino acid phenylalanine. This was followed by experiments in the laboratory of Severo Ochoa demonstrating that the poly-adenine RNA sequence (AAAAA.) coded for the p

7、olypeptide, poly-lysine. and the poly-cytosine RNA sequence (CCCCC.) coded for the polypeptide, poly-proline. Therefore the codon AAA specified the amino acid lysine, and the codon CCC specified the amino acid proline.,Using different copolymers most of the remaining codons were then determined. Ext

8、ending this work, Nirenberg and Philip Leder revealed the triplet nature of the genetic code and allowed the codons of the standard genetic code to be deciphered. In these experiments various combinations of mRNA were passed through a filter which contained ribosomes, the components of cells that tr

9、anslate RNA into protein. Unique triplets promoted the binding of specific tRNAs to the ribosome. Leder and N,Subsequent work by Har Gobind Khorana identified the rest of the genetic code. Shortly thereafter, Robert W. Holley determined the structure of transfer RNA (tRNA), the adapter molecule that

10、 facilitates the process of translating RNA into protein. This work was based upon earlier studies by Severo Ochoa, who received the Nobel prize in 1959 for his work on the enzymology of RNA synthesis. In 1968, Khorana, Holley and Nirenberg received the Nobel Prize in Physiology or Medicine for thei

11、r work.,1961年，Howard Dintzis证明血红蛋白肽链合成方向是从N-末端向C-末端进行。,19731976年，麦胚无细胞体系、兔网织无细胞体系分别建立，蛋白质合成的具体过程终于揭晓。,The ribosome, shown at the right, is a factory for the manufacture of polypeptides. Amino acids are carried into the ribosome, one at a time, connected to transfer RNA molecules (blue). Each amino a

12、cid is joined to the growing polypeptide chain, which detaches from the ribosome only once it is completed. This assembly line approach allows even very long polypeptide chains to be assembled rapidly and with impressive accuracy.,Protein Assembly,2009年因“核糖体的结构和功能”的研究而获得2009年的诺贝尔化学奖。,Venkatraman Ram

13、akrishnan, Thomas A. Steitz and Ada E. Yonath,上两章我们学习了DNA 和 RNA 的合成. 我们这章主要学习protein 合成的机制, 也叫蛋白质的翻译。,主要发现：,上两章我们学习了DNA 和 RNA 的合成. 我们这章主要学习protein 合成的机制, 也叫蛋白质的翻译。,主要发现：,Translation,第 一 节,20种氨基酸(AA)作为原料 酶及众多蛋白因子，如IF、eIF ATP、GTP、无机离子,参与蛋白质生物合成的物质包括,三种RNA mRNA（messenger RNA, 信使RNA） rRNA（ribosomal RNA,

14、 核蛋白体RNA） tRNA（transfer RNA, 转移RNA）,一、翻译模板mRNA及遗传密码,mRNA是遗传信息的携带者,遗传学将编码一个多肽的遗传单位称为顺反子(cistron)。 原核细胞中数个结构基因常串联为一个转录单位，转录生成的mRNA可编码几种功能相关的蛋白质，为多顺反子(polycistron) 。 真核mRNA只编码一种蛋白质，为单顺反子(single cistron) 。,原核生物的多顺反子,真核生物的单顺反子,目 录,mRNA上存在遗传密码,mRNA分子上从5至3方向，由AUG开始，每3个核苷酸为一组，决定肽链上某一个氨基酸或蛋白质合成的起始、终止信号，称为三联体

15、密码(triplet coden)。,起始密码(initiation coden): AUG,终止密码(termination coden): UAA，UAG，UGA,遗传密码表,目 录,从mRNA 5端起始密码子AUG到3端终止密码子之间的核苷酸序列，各个三联体密码连续排列编码一个蛋白质多肽链，称为开放阅读框架(open reading frame, ORF)。,1. 连续性(commaless),遗传密码的特点,编码蛋白质氨基酸序列的各个三联体密码连续阅读，密码间既无间断也无交叉。,基因损伤引起mRNA阅读框架内的碱基发生插入或缺失，可能导致框移突变(frameshift mutation

16、)。,蛋白质的生物合成，即翻译，就是将核酸中由 4 种核苷酸序列编码的遗传信息，通过遗传密码破译的方式解读为蛋白质一级结构中20种氨基酸的排列顺序 。,2. 简并性(degeneracy),遗传密码中，除色氨酸和甲硫氨酸仅有一个密码子外，其余氨基酸有2、3、4个或多至6个三联体为其编码。,目 录,2. 简并性(degeneracy),目 录,3. 通用性(universal),蛋白质生物合成的整套密码，从原核生物到人类都通用。 已发现少数例外，如动物细胞的线粒体、植物细胞的叶绿体。 密码的通用性进一步证明各种生物进化自同一祖先。,4. 摆动性(wobble),转运氨基酸的tRNA的反密码需要通

17、过碱基互补与mRNA上的遗传密码反向配对结合，但反密码与密码间不严格遵守常见的碱基配对规律，称为摆动配对。,U,摆动配对,目 录,原核生物翻译过程中核蛋白体结构模式:,A位：氨基酰位 (aminoacyl site),P位：肽酰位 (peptidyl site),E位：排出位 (exit site),目 录,tRNA的三级结构示意图,密码子、反密码子配对的摆动现象,Protein Assembly. The ribosome, shown at the right, is a factory for the manufacture of polypeptides. Amino acids ar

18、e carried into the ribosome, one at a time, connected to transfer RNA molecules (blue). Each amino acid is joined to the growing polypeptide chain, which detaches from the ribosome only once it is completed. This assembly line approach allows even very long polypeptide chains to be assembled rapidly

19、 and with impressive accuracy.,核蛋白体是多肽链合成的装置,目 录,不同细胞核蛋白体的组成,核蛋白体的组成,目 录,Ribosomes at low resolution: Election micrographs of (A) 30S subunits, (B) 50S subunits, and 70S ribosomes,一 、蛋白质合成需要将核苷酸序列翻译为氨基酸序列,1、Key points:1.1 蛋白质合成在生物界是一样的 1.2 合成有方向性，从amino-to carboxyl (N-C端)1.3 aminoacyl-tRNA synthetas

20、e是蛋白质合成的关键酶（氨基酰-tRNA合成酶）,1.4. The synthesis of long proteins requires a low error frequency（少出错）,2、Key players in protein synthesis2.1 Transfer RNA,Transfer RNA (tRNA),Robert Holly经过7年努力在1965年搞清楚了Yeast 的Alanine-tRNA序列。随后的研究表明其它的tRNA也有类似的结构，具体如下： 单链含有73-93个核糖核苷酸 有7-15个特殊碱基（甲基化等修饰） 有差不多一半的碱基自我配对形成双螺旋结

21、构，但有下列例外：3CCA末端，是连接氨基酸位点。（其它详见第56页） 5端tRNA磷酸化，通常是pG. 活化的氨基酸与3CCA末端连接 反义密码子定位于中心附近并形成环状结构,2.1. 1. Transfer RNA Molecules have a common Design (tRNA结构有一致性),蛋白质合成的保真性意味着对mRNA上的三联密码有严格的识别 氨基酸本身不能识别三联密码子，但tRNA能够识别三联密码子，且遵循Watson-Crick碱基配对规则 tRNA起着氨基酸运载工具和蛋白质生物合成适配器的作用,2.2.2. The activated Amino Acid and

22、the Anticodon of tRNA Are at Opposite Ends of the L-Shaped Molecule,The four helices of the secondary structure of tRNA stack to form an l-shaped structure,The archtecture of the tRNA molecule is well suited to its role as adaptor： the anticodon is available to interact with an appropriate codon on

23、mRNA while the end that is linked to an activated amino acid is well positioned to participate in peptide-bond formation,2.2.3. Aminoacyl-transfer RNA synthetases read the genetic code,（氨基酰-tRNA合成酶能够识别遗传密码）,为什么？,氨基酸分子与tRNA结合非常关键的原因有两点：某一特定的氨基酸与一特异的tRNA结合反映了遗传密码子的作用两个氨基酸的结合是一个耗能的过程,2.2. 4. 氨基酸活化形成氨基酰

24、-tRNA,氨基酰-tRNA合成酶(aminoacyl-tRNA synthetase),氨基酸的活化,第一步反应,氨基酸 ATP-E 氨基酰-AMP-E AMP PPi,目 录,第二步反应,氨基酰-AMP-E tRNA 氨基酰-tRNA AMP E,目 录,2.2. 5. 氨基酰-tRNA合成酶(aminoacyl-tRNA synthetase)的活性部位有选择性地结合氨基酸,实际上Threonine, Valine and Serine 的结构非常类似，但threonyl-tRNA synthase仍然将它们区分开,The structure of a large fragment of

25、 threonyl-tRNA synthetase reveals that the amino acid-binding site includes a zinc ion that coordinates threonine through its amino and hydroxyl groups. Only one subunit of the dimeric enzyme is shown,2.2. 6. Proofreading by 氨基酰-tRNA合成酶(aminoacyl-tRNA synthetase)increase the fidelity of protein synt

26、hesis,The results of mutagenesis studies revealed the position of the editing site (shown in green) in threonyl-tRNA synthetase,The flexible CCA arm of an aminoacyl-tRNA can move the amino acid between the activation site and the editing site. If the amino acids fits well into the editing site, the

27、amino acid is removed by hydrolysis,2.2. 7. 氨基酰-tRNA合成酶(aminoacyl-tRNA synthetase)能识别tRNA分子的Anticodon Loops and Acceptor Stems,Threonyl-tRNA synthetase complex. The structure of the complex between threonyl-tRNA synthetase and tRNAThr reveals that the synthetase binds to both the acceptor stem and t

28、he anticodon loop,Glutaminyl-tRNA synthetase complex. The structure of this complex reveals that the synthetase interacts with base pair G10:C25 in addition to the acceptor step and anticodon loop,2.2.8. 氨基酰-tRNA合成酶 (aminoacyl-tRNA synthetase) 有两大类,2.3. Ribosomal RNAs(5S, 16S, and 23S rRNA) play a c

29、entral role in protein synthesis,Ribosomal RNA folding pattern The secondary structure of 16S ribosomal RNA,B. The tertiary structure of 16S RNA determined by x-ray crystallography.,Ribosomal Protein structure. The structure of ribosomal protein L19 of the 50S ribosomal subunit reveals a long segmen

30、t of extended structure that fits through some of the cavities within the 23S RNA molecule.,Formylation of methionyl-tRNA,2.4 Protein are synthesized in the Amino-to Carboxyl Direction,2.5 Messenger RNA is translated in the 5-to-3 Direction,2.6 The start Signal is AUG (or GUG) Preceded by Several Ba

31、ses that Pair with 16S rRNA,Two kinds of interaction determine where protein synthesis start,Initiation sites. Sequences of mRNA initiation sites of protein synthesis in some bacterial and viral mRNA molecules. Comparison of these sequences reveals some recurring feature,2.7 Bacterial protein synthe

32、sis is initiated by Formylmethionyl transfer RNA,2.8 Ribosomes have three tRNA-Binding sites that bridge the 30S and 50S subunits,翻译后的折叠和加工修饰Folding and PosttranslationalProcessing,第三节,从核糖体释放出的新生多肽链不一定是具备生物活性的成熟蛋白质，必需经过各种翻译后修饰、加工过程才转变为有活性的成熟蛋白。,主要包括：,蛋白质折叠（protein folding） 翻译后加工（post-translation pro

33、cessing）,一、新生肽链经历翻译后修饰,（一）肽链N端和C端的切除和/或化学修饰,细胞内的脱甲酰基酶或氨基肽酶可以去除N-甲酰基、N-末端蛋氨酸或N-末端的一段肽链。,在真核细胞，50%的蛋白质在翻译后，氨基末端的氨基发生乙酰化。,有些情况下，羧基末端的残基也可被酶切除。,（二）水解加工包括多蛋白水解加工和内含肽的剪接,1多蛋白水解加工可产生多种肽链,一些蛋白质在合成之初是含有一系列头尾相连的蛋白质的长多肽链。多肽链的水解将裂解释放出各种蛋白质，释放出的蛋白质可能具有完全不同的功能，这些蛋白质称为多蛋白。,阿皮素原(POMC)的水解加工：,2内含肽切除导致外显肽连接,内含肽是蛋白质的内部

34、片段，翻译后很快被剪切，两个外显肽连接到一起。内含肽的长度一般在300 600个氨基酸之间。 剪接是由内含肽自我催化的。,1. 个别氨基酸可进行甲基化和乙酰化修饰 2. 蛋白质糖基化（glycosylation）修饰是一种 复杂的化学修饰 3. 某些蛋白质加入异戊二烯基团 4. 结合蛋白质加入辅基 5. 大多数蛋白质有二硫键的形成,（三）氨基酸残基的化学修饰有多种形式,二、折叠是肽链高级结构形成的过程,第一种模式认为是按等级进行的 ：新生肽链随着序列的不断延伸按等级逐步折叠，产生正确的二级结构、模序直至形成结构域和多肽链。 第二种模式：多肽链通过非极性残基间疏水作用介导,自动折叠成 “熔球（m

35、olten globule)”的紧密结构。 细胞内大多数天然蛋白质折叠需要一些辅助性蛋白。,（一）多肽链通过分步反应快速地进行折叠,核糖体结合的分子伴侣： 触发因子（trigger factor，TF） 新生链相关复合物（nascent chain-associated complex，NAC） 非核糖体结合的分子伴侣： 70kD/40kD热休克蛋白（heat shock protein, Hsp）家族 60kD/10kD热休克蛋白家族 蛋白质二硫键异构酶 肽酰脯氨酸异构酶,1分子伴侣有核糖体结合和非核糖体结合两类,（二）分子伴侣参与蛋白质折叠,细胞内分子伴侣完成以下功能：,封闭待折叠蛋白暴露的疏水区段； 创建一个隔离的环境，蛋白质可互不干扰在此折叠； 促进折叠和去聚合； 遇到应激，使已折叠的蛋白质去折叠。,（1）热休克蛋白70(Hsp70)的两个主要功能域为折叠所必需,还需要辅助因子Hsp40和Grp E,非核糖体结合的分子伴侣,Hsp70反应循环,（2）大肠杆菌的Gro EL属于热休克蛋白60（Hsp60）家族,需要辅分子伴素10(cochaperonin 10) Gro ES的帮助,（3） 二硫键异构酶催化链内