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姓名	瑞絲娜(Gita Riswana Nawung Rida)  查詢紙本館藏	畢業系所	生命科學系
論文名稱	(Recognition of a non-canonical tRNAAla by a non-canonical alanyl-tRNA synthetase)
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摘要(中)	Abstract (in Chinese) Aminoacyl-tRNA synthetase (aaRSs) is a group of essential enzymes whose main function is to connect specific amino acids to their corresponding tRNAs to participate in protein synthesis. AlanyltRNA synthetase (AlaRS) contains two highly conserved amino acid residues, N and D, which are primarily used to identify G3:U70 recognition determinants on tRNAAla. Surprisingly, these two residues were replaced by G and E in Caenorhabditis elegans mitochondrial AlaRS (CeAlaRSm). To elucidate the mechanism of tRNAAla recognition, we performed point mutations on these two residues and analyzed their effects on enzyme activity. We used E. coli microhelixAla as a receptor for the amine acylation reaction. Our results showed that the G322 mutation A or N had little effect on the activity of its complementary yeast AlaRS rejecting strains, and that G322A or G322N could effectively provide the AlaRS activity required for the growth of the rejected strains; The E420A and E420D mutations are similar. Amine acylation experiments have shown that these mutations do not impair the amine alkylation activity of CeAlaRSm, and both wild-type and mutant enzymes can effectively amide amichelixAla containing G3:U70. Although wild enzymes cannot aminoimide mutate microhelixAla (containing G3:C70 or A3:U70), mutant enzymes can still effectively aminoiminate mutant microhelixAla. These results suggest that CeAlaRSm uses negative interaction to screen non-G3:U70 tRNAs.
摘要(英)	Aminoacyl-tRNA synthetases (aaRSs) belong to a group of essential enzymes that are involved in protein synthesis. AaRSs attach a specific amino acid to its cognate tRNA, forming aa-tRNA, which is then delivered to ribosomes for polypeptide synthesis. Alanyl-tRNA synthetase (AlaRS) contains two highly conserved amino acid residues in its tRNA-recognition domain, Asparagine (N) and Aspartic acid (D), that play a role in recognizing the universal identity element of tRNAAla (G3:U70). Surprisingly, these two conserved residues are respectively substituted by G322 and E420 in the Caenorhabditis elegans mitochondrial AlaRS (CeAlaRSm). To elucidate the mechanism underlying the specific tRNA recognition, we mutated these two residues and analyzed their effects on the enzyme’s activities. In our assay, we used E. coli microhelixAla as the substrate for aminoacylation. Our functional assay showed that mutation of G322 to Alanine (A) or Asparagine (N) had little effect on its activity to rescue a yeast mitochondrial AlaRS knockout strain on YPG. A similar scenario was observed for E420A and E420D mutants. Aminoacylation assay toward wild-type E. coli microhelixAla further showed that these mutations had little effect on the charging activity of CeAlaRSm. The wild-type and mutant CeAlaRSm enzymes showed a comparable activity towards the wild-type microhelixAla with G3:U70. While the wild-type enzyme failed to charge the mutant microhelixAla (with G3:C70 or A3:U70), the mutant enzyme retained a full charging activity towards these two mutant microhelicesAla. These data suggest that G322 and E420 of CeAlaRSm recognizes G3:U70 through negative interaction; they are used for selection against non-G3:U70 tRNA.
關鍵字(中)	★ Alanyl-tRNA synthetase (AlaRS) ★ Aminoacylation ★ Identity element ★ Protein synthesis	關鍵字(英)	★ Alanyl-tRNA synthetase (AlaRS) ★ Aminoacylation ★ Identity element ★ Protein synthesis
論文目次	TABLE OF CONTENT Abstract (in Chinese) i ABSTRACT ii ACKNOWLEDGEMENT iii TABLE OF CONTENT iv LIST OF FIGURES vi ABBREVIATION vii CHAPTER I INTRODUCTION 1 1.1 Aminoacyl-tRNA synthetases (aaRSs) are key enzymes for protein synthesis 1 1.2 Alanyl-tRNA synthetase (AlaRS) possesses a prototype structure 2 1.3 tRNAAla identity is established through recognition of the G3:U70 pair by AlaRS 3 1.4 Caenorhabditis elegans possesses a non-canonical mitochondrial alanyl-tRNA synthetase and a non-canonical tRNAAla 3 1.5 Aim of Study 4 CHAPTER II MATERIALS and METHODS 5 2.1 Plasmids construction and protein purification of CeAlaRSm 5 2.2 In vivo heterologous complementation assay 6 2.3 Western Blotting 7 2.4 Substrate preparation 7 2.5 In vitro Aminoacylation assay 8 CHAPTER III RESULTS 9 3.1 Mutagenesis of G322 and E420 9 3.2 CeAlaRSm mutants could rescue a yeast ALA1 knockout strain on YPG 9 3.3 Expression of mutants CeAlaRSm in E. coli 10 3.4 Aminoacylation of WT microhelixAla by G322A and E420A 11 3.5 Aminoacylation of mutant microhelicesAla by G322A and E420A 12 CHAPTER IV DISCUSSION 13 4.1 CeAlaRSm use Gly (G) and Glu (E) residues to select against non-cognate tRNA 13 4.2 Mitochondrial AaRSs coadaptation with its cognate mttRNA 14 LIST OF FIGURES 17 REFERENCES 24 APPENDIX A PRIMER LIST 28 APPENDIX B PLASMID LIST 30 APPENDIX C SUPPLEMENTARY FIGURES 33
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指導教授	王健家(Chien-Chia Wang)	審核日期	2022-7-19
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