CHAPTER4 Translation

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1 CHAPTER4 Translation 4.1 Outline of Translation 4.2 Genetic Code 4.3 trna and Anticodon 4.4 Ribosome 4.5 Protein Synthesis 4.6 Posttranslational Events

2 4.1 Outline of Translation

4 From mrna to protein Pre-mRNA Mature mrna Cap 5 uncoding region AUG Splicing and processing UAG Coding region polya 3 uncoding region Pre-protein Mature protein Translation Posttranslational modification, processing and folding

5 4.2 Genetic Code Protein: 20 different amino acids mrna: 4 different bases A single base as a codon: 4 codons Pairs of bases as codons: 16 codons Triplets of bases as codens: 64 codons The genetic code is the correspondence between base sequences in DNA (or RNA) and amino acids in protein. A codon is a triplet of nucleotides that represents an amino acid or a start/termination signal of translation.

6 The genetic code is triplet

8 终止密码子 :UAA( 赭石, ochre); UAG( 琥珀, amber), UGA( 蛋白石, opal) 起始密码子 : AUG ( 有些为 GUG)

Open reading frame An open reading frame (ORF) is a sequence of DNA consisting of triplets that can be translated into amino acids starting with an initiation codon and ending with

9 Open reading frame An open reading frame (ORF) is a sequence of DNA consisting of triplets that can be translated into amino acids starting with an initiation codon and ending with a termination codon. A reading frame is one of the three possible ways of reading a nucleotide sequence. Each reading frame divides the sequence into a series of successive triplets.

10 There are three possible ways of translating any nucleotide sequence into protein, depending on the starting point. For example: For the sequence ACGACGACGACGACGACG the three possible reading frames are: (AUG) ACG ACG ACG ACG ACG ACG ACG.. (AUG) CGA CGA CGA CGA CGA CGA CGA.. (AUG) GAC GAC GAC GAC GAC GAC GAC..

11 遗传密码的特点 1. 遗传密码的简并性简并性 (degeneracy): 指一个氨基酸有一个以上的密码子为其编码 2. 在 mrna 模板上的密码子是连续的, 在前一个密码子与后一个密码子之间没有间隔 4. 同一氨基酸的不同遗传密码的使用频率不同 5. 遗传密码的通用性和特殊性遗传密码对病毒, 细菌, 动物和植物而言都适用, 但是也存在特定条件下的一些例外支原体 :UAG 编码色氨酸 ; 嗜热四膜虫 :UAA 编码谷氨酰胺 ; 线粒体 DNA 与核 DNA 的密码子存在差异

13 4.3 trna and Anticodon

14 trna 结构 1 接受臂 CCA 末端氨基酸结合位点 2 TϕC 臂 / 环 ϕ=pseudouridine 假尿嘧啶 3 多余臂 (extra arm) 4 反密码子臂 / 环 5 D 臂 /DHU 环 DHU=dihydrouridine 二氢尿嘧啶

16 Wobble hypothesis Codon-anticodon recognition involves wobbling: A trna recognizes more than one codon by unusual (non-g C, non-a U) pairing with the third base of a codon.

17 Wobble hypothesis

18 Wobble hypothesis

19 Aminoacyl-tRNA synthetases Aminoacyl-tRNA synthetases are enzymes that charge trna with an amino acid to generate aminoacyl-trna in a two-stage reaction that uses energy from ATP. Step 1:Amino acid + ATP Aminoacyl-AMP + PPi Step 2:Aminoacyl-AMP + trna Aminoacyl- trna + AMP Total:Amino acid +ATP + trna Aminoacyl- trna+amp +PPi

20 There are 20 aminoacyl-trna synthetases in each cell. Each charges all the trnas that represent a particular amino acid. Recognition of a trna is based on a small number of points of contact in the trna sequence.

21 4.4 Ribosome

22 Assembly map for the 30S subunit

23 The ribosome has several active centers

24 Three trna-binding sites in Ribosome An aminoacyl-trna enters the A site. Peptidyl-tRNA is bound in the P site. Deacylated trna exits via the E site. An amino acid is added to the polypeptide chain by transferring the polypeptide from peptidyltrna in the P site to aminoacyl-trna in the A site.

25 Shine-Dalgarno Sequence

26 4.5 Protein Synthesis Factors involved in protein synthesis The process of Protein synthesis Protein synthesis inhibition Transcription coupled translation in prokaryotic cell

27 4.5.1 Factors involved in protein synthesis 一 Initiation factor (IF, 起始因子 ) ( 一 )Prokaryotic Initiation factors 1 IF-1:IF-1 prevents trnas form binding to the portion of the small subunit that will bacame part of the A site. 2 IF-2:IF-2 is a GTPase which facilitates the association of fmettrna fmet with the small subunite by forming the IF2 GTP fmettrna fmet complex. 3 IF-3:IF-3 binds to the 30S small subunit and block it from reassociating with a 50S large subunit.

28 ( 二 )Eukaryotic Initiation Factors More than 10 IFs: eif1, eif2, eif2b, eif3, eif4a, eif4b, eif4c eif4d, eif4e, eif5 eif2 最重要, 与 GTP Met- trna i Met 生成三元复合物 eif2 GTP Met- trna i Met eif3 的作用类似于原核生物的 IF3

29 二 Elongation Factor (EF, 延伸因子 ) ( 一 )Prokaryotic Elongation Factors 1 EF-T U With the presence of GTP, EF-T U GTP can interact with aminoaclytrna to form a stable EF-T U GTP AA- trna complex, and bring the AA-tRNA into the A site according to the genetic code on mrna. 2 EF-T S EF-T S facilitates the conversion from EF-T U GDP to EF-T U GTP. 3 EF-G EF-G is a GTPase responsible for the translocation of the peptidyltrna form A site to P site. ( 二 )Eukaryotic Elongation Factors Two EFs:eEF-1 和 eef-2 eef1α has a function similar with that of prokaryotic EF-T U ; eef1βγ has a function similar with that of prokaryotic EF-T S ; eef-2 has a function similar with that of prokaryotic EF-G

30 三 Release Factor (RF, 释放因子 ) RFs recognize the stop codons on mrna, terminate the protein synthesis and stimulates the polypeptide release 1. Prokaryotic RFs:RF 1 RF 2 RF 3 Class I : RF 1 that recognizes UAA and UAG RF 2 that recognizes UAA and UGA Class II: RF 3 that triggers the hydrolysis of peptidyl-trna linkage by Class I RF, and help to remove Class I RFs from ribosome. 2. Eukaryotic RF: erf1, erf1 recognizes all of the three stop codons UAA UAG UGA.

31 4.5.2 The process of protein synthesis I. Aminoacyl-tRNA Charging II. Initiation of peptide synthesis III. Elongation * Binding of aminoacyl-trna to Ribosome * Peptidyl transferase reaction * Translocation IV. Termination of peptide synthesis and peptide release

32 Aminoacyl-tRNA Charging Aminoacyl-tRNA synthetases Step 1: Amino acid + ATP Aminoacyl-AMP + PPi Step 2:Aminoacyl-AMP + trna Aminoacyl- trna + AMP Total:Amino acid +ATP + trna Aminoacyl- trna+amp +PPi

34 Charging of the Initiation trna ----trnafmet

35 Prokaryotic translation initiation

36 Eukaryotic translation initiation 真核至少有 10 个因子直接或间接参与肽链合成的起始起始因子在 mrna 5 端形成 cap-binding complex; 在 GTP, eif2 eif2b 和 eif3 作用下, Met-tRNA i Met 与 40S 小亚基结合, 形成 43S 复合物 ; 43S 复合物结合到 mrna 5 端, 形成 48S 复合物, 从 5 到 3 沿 mrna 滑动至 AUG; GTP 水解, 起始因子释放,eIF5B 介导 60S 大亚基的结合

38 Elongation Three steps are performed to add one amino acid in to the polypeptide: Step 1. Binding of aminoacyl-trna to Ribosome A site Step 2. Peptidyl transferase reaction Step 3. Translocation

39 Step 1. Binding of aminoacyl-trna to Ribosome A site Only fmet-trna fmet can be used for initiation by 30S subunits; only other aminoacyl-trnas (AA-tRNA) can be used for elongation by 70S ribosomes.

41 Step 2. Peptidyl transferase reaction Peptidyl Transferase center The Peptidyl Transferase center of the ribosome is composed entirely of RNA.

42 Step 3. Translocation

43 EF-TG

45 Termination of peptide synthesis and peptide release Peptide synthesis is terminated when the stop codon (UAA, UAG or UGA) is located at the ribosomal A site. There is no corresponding aminoacyl-trna to recognize the stop codon; Class I RFs (RF-1 and RF-2) take the A site through the interaction with stop codon, and release the peptide from the peptidyl-trna at the P site with assistant of Class II RF (RF-3). RRF (ribosome recycling factor) cooperates with EF-G and IF3 to separate the large and small ribosome subunits, release trna and mrna.

47 4.5.3 Protein synthesis inhibition Puromycin: terminates translation by mimicing a aminoacyl-trna in the A-site. Chloramphenicol:Inhibits the peptidyl transfer reaction. Tetracycline:Inhibits aminoacyl-trna binding to the A-site Erythromycin: Blocks exit of the growing polypeptide chain from ribosome; arrests translation. Hygromycin B: Prevents translocation of A-site trna to P-site. Diptheria Toxin: Inhibits EF-Tu function Cycloheximide: Inhibits peptidyl transferase activity of the 60s subunit.

49 4.5.4 Transcription coupled translation in prokaryotic cell In Eukaryotic cell, translation and transcription are performed separately in deferent regions of the cell; In prokaryotic cell, translation is coupled with transcription. Translation of mrna happens as soon as the mrna is produced by RNA polymerase.

50 The lac operon mrna

51 Polyribosome ( 多核糖体 )

52 4.6 Posttranslational Events Protein folding and processing Protein translocation Protein degradation

53 4.6.1 Protein folding and processing 1. Removal of the N-terminal fmet; 2. Removal of the nonfunctional segments; 3. Modification of amino acids such as methylation, acetylation, and phosphorylation. 4. Disulfide bonds formation between deferent peptide chains or in the same peptide chain. 分子伴侣 (molecular chaperone) 折叠酶 (foldase)

55 4.6.2 Protein translocation

56 Co-translational transport ( 共翻译转运 ) SRP (signal recognition particle );

57 Signal peptide

58 Post-translational transport ( 翻译后转运 ) I. Mitochondrial protein transport II. Nuclear protein transport

59 I. Mitochondrial protein transport A multipart leader contains signals that function in a hierarchical manner

60 II. Nuclear protein transport 核定位信号 (nuclear localization signal, NLS) 1. There is no apparent conservation of sequence of NLS signals; 2. Many NLS sequences take the form of a short, rather basic stretch of amino acids.

61 4.6.3 Protein degradation Prokaryotic cell: In E. coli, protein degradation is mainly through the ATP-dependent protease. Eukaryotic cell: Ubiquitin ( 泛素 ) mediated protein degradation Ubiquitin has a highly conserved sequence of 76 amino acids. It is linked via its COOH group to the ε NH 2 group of a lysine residue in a target protein

62 Ubiquitin Cycle The ubiquitin cycle involves three activities. E1 is linked to ubiquitin. E3 binds to the substrate protein. E2 transfers ubiquitin from E1 to the substrate. Further cycles generate polyubiquitin. in which each additional ubiquitin is added to the Lys at position 46 of the preceding ubiquitin. The formation of polyubiquitin is a signal for the proteasome to degrade the protein

64 Differences between eubacteria and eukaryotes Bacteria Ribosome: 30S+50S 70S Few initiation factors: IF-1, IF-2, IF-3 Elongation factors EF-Tu, EF-Ts, EF-G Release factors RF-1, RF2, RF3 Ribosome recycling factor RRF mrna is not capped Direct binding of 30S particle next to initiation codon (AUG) at Shine-Dalgarno sequence, 5 - AGGAGGU-3 Translation coupled to transcription There are polycistron mrna Eukaryotes Ribosome: 40S+60S 80S Many initiation factors eif1, eif1a, eif2, eif2b, eif3, eif4a, eif4b, eif4e, eif4f, eif4g, eif4h, eif5, eif5b, eif6 Elongation factors eef1, eef2 Release factors erf1, (erf3) Most mrna is capped at 5 end and polyadenylated at 3 end 43S particle is recruited to 5 cap structure, and then scans from 5 to 3 to find the initiation codon(aug) Translation in cytoplasm apart from transcription Single-cistron mrna

65 Summary Genetic code; Open reading frame; Wobble hypothesis; Cognate trna(isoaccepting trna); Shine-Dalgarno sequence; Molecular chaperone; Signal sequence(signal peptide);ubiquitin 遗传密码的特点 ; trna 的结构 ; 摆动假说 ; 核糖体的组成及其活性位点 ; 简述原核蛋白质 ( 多肽 ) 生物合成的过程 ; 真核和原核生物在蛋白质生物合成上的差异 ; 抑制蛋白合成的抗生素及其作用机理 ; 蛋白质前体加工和修饰的主要方式 ; 共翻译转运和翻译后转运 ; 泛素及其在蛋白质降解中的作用

CHAPTER4 Translation

CHAPTER4 Translation 4.1 Outline of Translation 4.2 Genetic Code 4.3 trna and Anticodon 4.4 Ribosome 4.5 Protein Synthesis 4.6 Posttranslational Events 4.1 Outline of Translation From mrna to protein