CHAPTER4 Translation

Transcription

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

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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

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8 UAA(, ochre); UAG(, amber), UGA(, opal) : AUG ( GUG)

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 1. (degeneracy): 2. mrna 4. 5.,, UAG UAA DNA DNA

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13 4.3 trna and Anticodon

14 trna 1 CCA 2 TϕC / ϕ=pseudouridine 3 (extra arm) 4 / 5 D /DHU DHU=dihydrouridine

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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 Initiation factor (IF, ) 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 Eukaryotic Initiation Factors More than 10 IFs eif1, eif2, eif2b, eif3, eif4a, eif4b, eif4c eif4d, eif4e, eif5 eif2 GTP Met- trna i Met eif2 GTP Met- trna i Met eif3 IF3

29 Eukaryotic Elongation Factors Two EFs eef-1 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 Elongation Factor (EF, ) 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.

30 Release Factor (RF, 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

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34 Charging of the Initiation trna ----trnafmet

35 Prokaryotic translation initiation

36 Eukaryotic translation initiation 10 mrna 5 cap-binding complex GTP, eif2 eif2b eif3, Met-tRNA i Met 40S, 43S 43S mrna 5 48S 5 3 mrna AUG GTP eif5b 60S

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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.

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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

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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.

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47 Cycloheximide: Inhibits peptidyl transferase activity of the 60s subunit 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

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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 ( )

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. (molecular chaperone) foldase

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55 4.6.2 Protein translocation

56 Co-translational transport ( ) SRP (signal recognition particle );

57 Signal peptide

58 Post-translational transport ( ) 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 (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 ( ) mediated protein degradation Ubiquitin has a highly conserved sequence of 76 amino acids. It is linked via its COOH group to the 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

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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 trna