《生物化学》课程PPT教学课件（英文版）chapter 27（first part）protein metabolism

18. The anticodon and/or the amino acid arms of a tRNA are key for a specific aminoacyl-tRNa synthetase to recognize This was revealed via: crystal structure determination of the synthetases complexed with their cognate tRNAS, sequence comparison of trnas binding to the same synthetase and mutagenesis studies When the anticodon of tRNAVal is changed from UaC to cau. the mutated tRNAVal can be recognized by Met-tRNa synthetase and generating a met-tRNAvVal

18. The anticodon and/or the amino acid arms of a tRNA are key for a specific aminoacyl-tRNA synthetase to recognize • This was revealed via: crystal structure determination of the synthetases complexed with their cognate tRNAs; sequence comparison of tRNAs binding to the same synthetase; and mutagenesis studies. • When the anticodon of tRNAVal is changed from UAC to CAU, the mutated tRNAVal can be recognized by Met-tRNA synthetase and generating a Met-tRNAVal

When the 3: 70 base pair on the amino acid arm of tRNacys is changed from from CG to G-U. the mutated tRNACys is able to carry Ala, instead of Cys a"microhelix' containing only 24 of the 76 nucleotides of tRNAAla is recognized and aminoacylated by Ala-tRNa synthetase

• When the 3:70 base pair on the amino acid arm of tRNACys is changed from from C.G to G.U, the mutated tRNACys is able to carry Ala, instead of Cys. • A “microhelix” containing only 24 of the 76 nucleotides of tRNAAla is recognized and aminoacylated by Ala-tRNA synthetase

3 Amino acid o arm Positions(green and 5 orange)of nucleotides involved in recognition TUC arm DHU arm between tRNAs and aminoacyl-tRNA synthetases Extra arm Anticodon arm Anticodon

Positions (green and orange) of nucleotides involved in recognition between tRNAs and aminoacyl-tRNA synthetases

76 A single g=u base pair is the only element needed for specific binding of tRNAAla and aminoacylation by Ala-tRNA synthetase G●U70 3 76 60 10 5 1 G●U70 30 40 66 Deleted nucleotides 10 (a) (b)

A single G=U base pair is the only element needed for specific binding of tRNAAla and aminoacylation by Ala-tRNA synthetase

19 Proofreading by some aminoacyl- tRNA synthetases increases the fidelity of protein synthesis The identity of the amino acid attached to a specific tRNa is not checked by the ribosome(ala-tRNaCyS experiment) The calculated rate of incorrect incorporation of val in place of lle is I in 200, but the observed is only I in 3000. Some synthetases are able to proofread the incorrectly incorporated similar amino acids(e.g, between Val and Ile) at the aminoacyl-AMP stage or the aminoacyl tRNA stage

19. Proofreading by some aminoacyl￾tRNA synthetases increases the fidelity of protein synthesis • The identity of the amino acid attached to a specific tRNA is not checked by the ribosome (Ala-tRNACys experiment). • The calculated rate of incorrect incorporation of Val in place of Ile is 1 in 200, but the observed is only 1 in 3000. • Some synthetases are able to proofread the incorrectly incorporated similar amino acids (e.g., between Val and Ile) at the aminoacyl-AMP stage or the aminoacyl￾tRNA stage

There seems to be separate proofreading active site (s)on such synthetases In a few synthetases that activate amino acids having no close structural relatives. no such proofreading activities have yet identified(selected at the substrate binding level)

• There seems to be separate proofreading active site (s) on such synthetases. • In a few synthetases that activate amino acids having no close structural relatives, no such proofreading activities have yet identified (selected at the substrate binding level)

The codon on mRNA is recognized by the anticodon of tRNA rather than by the activated amino acid Cysteinyl-tRNA H O R H O Cysteine tRNACys synthetase HS-CH-C-C-0-tRNACYs nickel H一CH2-C-C-0-tRNA ATP+H,0 AMP+2 Pi NH Cys-tRNA YS Ala-tRNACY 1. Cys-tRNACyS is chemically converted to Ala-tRNACyS 2. Studies with in vitro protein synthesis systems proved that Ala-tRNA Cys will incorporate Ala at places of Cys using poly (G, t)as templates or hemoglobin mRNA as template (with [4Cl-Ala-tRNACys)

The codon on mRNA is recognized by the anticodon of tRNA rather than by the activated amino acid 1. Cys-tRNACys is chemically converted to Ala-tRNACys ; 2. Studies with in vitro protein synthesis systems proved that Ala-tRNACys will incorporate Ala at places of Cys: using poly(G,U) as templates or hemoglobin mRNA as template (with [14C]-Ala-tRNACys)

Some synthetases are able to Adenylation proofread the incorrectly incorporated amino acids Proofreading: aminoncyl-adernylate is hydrolyzed tRNA binding amino acid C At the aminoacyl- AMP stage hanging with tRNA Proofreading aminoacyl-tRNA is hydrolyzed Wrong amino acid a At the aminoacyl- ERNA Stage COOH Correct amino acid AMP Correctly Aminoacyl-tRNA charged tRNA

At the aminoacyl￾AMP stage At the aminoacyl￾tRNA stage Correctly charged tRNA Some synthetases are able to proofread the incorrectly incorporated amino acids

20. Met-tRNA Met recognizes the initiating AUG codon in almost all cells It was observed that about half of the n-terminal residues of proteins in E coli are met, and the n-terminal of nascent polypeptides is usually modified There are two tRNAs for recognizing AUg and Met in all organisms: one(tRNA Met) brings Met to the initiating AUG and the other(tRNA Met) brings met to internal AUgs The Met charged on tRNAMet(by Met-tRNA synthetase is specifically formylated by a transformylase to form fMet-tRNAMet in bacteria(Met-tRNA Met can not be formylated) fMet-tRNAtMet can only enter and only fMet-tRNAMet can enter the initiation site(site P)on the ribosome in

20. Met-tRNAi Met recognizes the initiating AUG codon in almost all cells • It was observed that about half of the N-terminal residues of proteins in E.coli are Met, and the N-terminal of nascent polypeptides is usually modified. • There are two tRNAs for recognizing AUG and Met in all organisms: one (tRNAi Met) brings Met to the initiating AUG and the other (tRNAMet) brings Met to internal AUGs. • The Met charged on tRNAi Met (by Met-tRNA synthetase) is specifically formylated by a transformylase to form fMet-tRNAfMet in bacteria (Met-tRNAMet can not be formylated). • fMet-tRNAfMet can only enter and only fMet-tRNAfMet can enter the initiation site (site P) on the ribosome in E.coli

Eukaryotes MetHtRNA tRNA Met+ Methionine Archaeans - Initiation fMetH tRNA Met Bacteria +CHO All cells tRNAMet+Methionine → MetHtRNA一→ Elongation Two types of methionine trNA (tRNA Met or tRNAMet in bacteria and tRNAMet are found in all cells Both trNas are recognized by the same Met-tRNA Synthetase

Two types of methionine tRNA (tRNAi Met or tRNAfMet in bacteria, and tRNAMet) are found in all cells. Both tRNAs are recognized by the same Met-tRNA synthetase