Protein Biosynthesis Translation( 翻译 )

1. Genetic information transfer------ Protein BiosynthesisTranslation(翻译) Lecturer: Jiao Li Phone: 021-65986142 Email: li2sister@163.com Lecturer: Jiao Li Phone: 021-65986142 Email: li2sister@163.com

2. Objectives After completion of this section, you should be able to : • List molecules (AminoacyltRNAsynthetase) involved in protein • Synthesis • 2. Describe the process of protein synthesis • 3.Post-translational processing and destination of newly • synthesized protein. • 4.Inhibitors of protein synthesis

3. Protein synthesis: using mRNA as the template, translate the nucleotide sequence of mRNA into the amino acid sequence of protein according to the genetic codon.

4. Replication Transcription Translation

5. Outline of the section System of protein biosynthesis Process of protein translation Post-translational modification Inhibitors of protein synthesis

6. Protein Biosynthesis System Section 1

7. Protein synthetic System • Direct template— mRNA • tRNA • Ribosomes ●20amino acids (AA) ●Enzyme and protein factors: IF、eIF、EF、RF ●ATP、GTP、Mg2+ Characteristics：complicated, accurate, dynamic,quick, energy consumed（90%）

8. §1.1 Genetic codon carrier-mRNA ● baseamino acid conversion：Genetic code a three-nucleotide codon in a nucleic acid (mRNA) sequence specifies a single amino acid, initiatiorcodon or terminator codon. This kind of three-nucleotide codon is called genetic codonor triplet codon. Initiator codon: AUG Terminator codon: UAA、UAG、UGA

9. Standard Genetic codon

10. Salient features Sense codon：code for amino acid — 61. Nonsense codon：code for no amino acid—terminator codon—UAA，UAG，UGA Initiator codon：as initiation signal for translation — AUG, GUG(prokaryote) .

11. ' ' U A A 3 A U 5 G A complete sequence of mRNA, from the initiation codon(AUG) to the termination codon, is termed as the open reading frame (ORF), which codes for the primary structure of polypeptide. UTR UTR

12. Properties of genetic code 1.Commaless • The genetic codons should be read continuously without spacing or overlapping. spacing × ※ Accuracy of start site × overlapping

13. Gene damage may causebase insertion or deletion on mRNA template, which leads to frame shift mutation. Insertion Deletion

14. 2.Sideness Reading frame：5’—3’ Translation ：5’—3’

15. 3.Degeneracy code1 amino acid (except Trp, Met) Code2/3/4/6 Providing multiple options for maintaining stabilization of species.

16. Number of genetic codon AA Number of genetic codon AA

17. 4.Universal • The genetic codons for amino acids are always the same.（basic reason of microbial infection） • With a few exceptions ： mitochondrial mRNA, chloroplast . Differences between mitochondrial codons and standard codons

18. §1.2 Amino acid carrier—tRNA/adaptor AA arm TCloop DHUloop Anticodon loop “Cloverleaf ” structure of tRNA

19. ●tRNAis the bridge between protein and genetic data tRNA 2 Amino acid Iso-tRNA tRNA 3/4/6 relative specificity of transferring

20. A—C—G mRNA —U—G—C Binding sites in tRNA Attachment site of aa DHUloop (binding rRNA in ribosome) Mathch with codons

21. 摆动性(wobble base pair) Non-Watson-Crick base pairing • the 5' base on the anticodon, which binds to the 3' base on the mRNA, was not as spatially confined as the other two bases, and could, thus, have non-standard base pairing.

22. wobble U

23. Wobble hypothesis 1 2 3 The first base of anticodon is key to determine the number of codons it can identify. G C U G C C mRNA: 5’ —G—C—A——3’ tRNA： 3’ —C—G—I——5’ Anticodon of ala from yeast 3 2 1

24. Diagram of wobble base pairing 5’base on anticodon3’base on codon A U C G U C G A G U U C A I

25. ●Activated amino acid —aminoacyl-tRNA Aminoacyl-tRNA：gly-tRNAgly Initiation aminoacyl-tRNA： (f)met-tRNAi(f)met Elongation aminoacyl-tRNA： ala-tRNAeala

26. §1.3 Protein synthesis site: （Ribosomes）

27. Components of ribosome

28. rRNA rRNA is important component in ribosome and plays a key role in ribosome assembling and functioning. 1. Maintaining structure of ribosome. Delete rRNA and ribosome will collapse. 2. Involved in binding to mRNA. 3. Key component in protein synthesis.

29. Illustration of protein synthesis in prokaryote Small subunit P site (Peptidylsite) D/donor site A site (Aminoacylsite) Acceptor site AA Nascent peptide chain Esite (Exit site) Large subunit

30. Function of ribosome in protein synthesis ① Containing protein-conducting channel ② Containing binding sites of IF, EF and RF ③ Containing three RNA binding sites， （ donor site, D 位); (peptidyl site , P 位）;（ acceptor site, aminoacylsite , A 位） ④ Peptidyltransferaseactivity ⑤GTPaseactivity

31. The Process of Protein Biosynthesis Section 2

32. Protein synthesis process Amino acid activation Ribosome cycle

33. Aminoacyl-tRNAsynthetase aa+ tRNA aminoacyl- tRNA ATP AMP＋PPi §2.1 Activation and transferration of amino acid a. Aminoacyl-tRNAsynthetase

34. Diagram of aminoacyl-tRNA synthetase tRNA ATP aminoacyl-tRNAsynthetase

35. Step 1 of reaction aa＋ATP-E —→ aminoacyl-AMP-E ＋ PPi ～ ～ A AA ～ A Aminoacyl-AMP Gain high energy

36. A Step 2 Adenosine Aminoacyl-AMP aminoacyl-AMP-E ＋ tRNA ↓ aminoacyl-tRNA ＋ AMP ＋ E Aminoacyl-tRNAsynthetase A Aminoacyl-tRNA Activation of aa and attachment to tRNA

37. Generation of amino-acyltRNA catalyzed by amino-acyltRNAsynthetase

38. b. Features of aminoacyl-tRNAsynthetase • 1. high specificity to substrate-amino acid. • 2. proofreading activity，second code system—tRNA。 Match correct aa to its corresponding tRNA

39. §2.2 Ribosome cycle（prokaryote） Protein synthesis initiates from AUG at 5’ end and decipher genetic codons to polymerize aa residues until stop codon occurs. • Initiation • Elongation • Termination Translation process： Major phases

40. Idetification of initiation site — SDsequence Prokaryote mRNA 5′ 3′ ORF UTR UTR Ribosomal binding site Initiation codon mRNA nt 16S-rRNA S-D sequence （ribosomal binding site）

41. 1. Initiation Translational initiation complex= mRNA + initiation aminoacyl-tRNA + ribosome

42. substrate+ initiation factor+ GTP + Mg2+

43. substrate+ initiation factor+ GTP + Mg2+ stageprofunction IF1 Binding small subunit 30S IF2Binding initiation amino-acyl- tRNAto AUG, GTPase IF3Binding small subunit 30S,facilitate the dissociation of ribosome

44. Formation of translational initiation complex in prokaryote （1）Ribosome acitivation （2）30S- initiation complex （3）70S- initiation complex

45. （1）Ribosome activation IF-1 IF-3

46. 5' 3' SD sequence A U G （2）30S-initiation complex a. mRNAbinds with small subunit of ribosome IF-1 IF-3

47. b. Initiation aminoacyl-tRNA( fMet-tRNAimet)binds to small subunit of ribosome IF-2 GTP 5' 3' SDsequence A U G Formyl-transferase IF-1 IF-3

48. 5' 3' SDsequence A U G （3）70S-initiation complex Large subunit binds to form 70s initiation complex IF-2 GTP Pi GDP IF-1 IF-3

49. IF-2 GTP 5' 3' SDsequence A U G IF-2 Pi -GTP GDP IF-3 IF-1

50. 2. Elongation The second and the next aminoacyl-tRNA in line bind to the ribosome along with GDP and elongation factor. • It consists of the following steps: • (entrance/registration) • (transpeptidation) • (translocation) Initiation complex+ elongation factor+ GTP + Mg2+