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姓名	黃美蘭(Dea Jolie Chrestella)  查詢紙本館藏	畢業系所	生命科學系
論文名稱	(tRNA aminoacylation by a naturally occurring mini-AlaRS)
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摘要(中)	丙胺酸-tRNA 合成酶 (Alanyl-tRNA synthetase, AlaRS) 屬於轉譯酵素中的一員，負責將丙胺酸 (Alanine) 轉移到其同源 tRNA上，形成 Ala-tRNAAla，然後將其遞送至核醣體為著蛋白質合成。AlaRS都有四個功能域，包含「催化結構域」、「tRNA 辨識結構域域」、「校正結構域」和「C-Ala結構域」。在AlaRS的tRNA 辨識結構域中有兩個高度保守的胺基酸殘基N及D (例如E. coli AlaRS的 N303 和 D400) 與 tRNAAla 中的接受柄上的G3:U70有專一性的交互作用。有趣的是，一種天然存在的mini-AlaRS被發現於卡氏棘阿米巴 (Acanthamoeba castellanii) 中的圖邦病毒 (Tupanvirus)，該酵素缺乏校正結構域和 C-Ala 結構域，並且在其 tRNA 辨識結構域中含有的是 P/T 而不是 N/D。親緣演化分析顯示，這種mini-AlaRS的親緣關係比起原核生物的AlaRS，與真核生物AlaRS的關係更緊密，體現出它起源於真核生物。胺醯化分析 (Aminoacylation assay) 顯示，儘管缺乏 N/D 胺基酸，但圖邦病毒的 AlaRS (TuAlaRS) 能夠專一地辨認含有G3:U70 的tRNAAla。然而奇怪的是，突變P/T卻對它的胺醯化活性或專一性沒有影響。另外，TuAlaRS能以相似的效率作用於microhelixAla；相對的，E. coli AlaRS偏好作用於tRNAAla過於microhelixAla。電泳遲緩分析更進一步顯示圖邦病毒AlaRS 與野生型 (G3:U70) 和突變型 (非 G3:U70) 的tRNAAla和microhelixAla 的結合有相似的親和度；而E. coli AlaRS偏向於與野生型tRNAAla結合。由於缺乏校正結構區域，因此圖邦病毒AlaRS相較於E. coli酵素，表現出更高機率的錯誤性胺醯化作用。本研究的重點在於這自然存在於mini-AlaRS的強力丙胺酸化活性。
摘要(英)	Alanyl-tRNA synthetase (AlaRS) belongs to a family of translation enzymes and is responsible for transferring alanine to its cognate tRNA, forming Ala-tRNAAla, which is then delivered to ribosomes for protein synthesis. AlaRS contains four functional domains: catalysis, tRNA-recognition, editing, and C-Ala domains. Two highly conserved amino acid residues N and D in the tRNA-recognition domain of AlaRS (N303 and D400 in E. coli AlaRS) make specific contacts with the identity element G3:U70 in the acceptor stem of tRNAAla. Interestingly, a naturally occurring mini-AlaRS was identified in the Tupanvirus of Acanthamoeba castellanii. This enzyme lacks the editing and C-Ala domains and contains P/T instead of N/D in its tRNA-recognition domain. Phylogenetic analysis indicated that this mini-AlaRS is more closely related to eukaryotic AlaRSs than to prokaryotic AlaRSs, suggesting its eukaryotic origin. Aminoacylation assay showed that Tupanvirus AlaRS specifically charges G3:U70-containing tRNAAla despite lacking the N/D residues. However, mutation of P/T had little effect on its aminoacylation activity or specificity. In addition, this enzyme could charge microhelixAla to a similar level. In contrast, E. coli AlaRS strongly preferred tRNAAla over microhelixAla. Electrophoretic mobility shift assay further showed that Tupanvirus AlaRS bound wild-type (G3:U70) and mutant (non-G3:U70) tRNAAla and microhelixAla with a similar affinity, while E. coli AlaRS preferentially bound WT tRNAAla. Due to the lack of the editing domain, Tupanvirus AlaRS exhibited a higher misacylation rate compared with the E. coli enzyme. This study highlights the strong alanylation activity of a naturally occurring mini-AlaRS.
關鍵字(中)	★ alanyl-tRNA synthetase ★ aminoacylation ★ giant virus ★ identity element ★ tRNA	關鍵字(英)	★ alanyl-tRNA synthetase ★ aminoacylation ★ giant virus ★ identity element ★ tRNA
論文目次	ABSTRACT i ABSTRACT (in Chinese) ii ACKNOWLEDGEMENT iii TABLE OF CONTENTS iv TABLE OF FIGURES v ABBREVIATION vii CHAPTER I INTRODUCTION 1 1.1.Aminoacyl-tRNA synthetases belong to a group of translation enzymes 1 1.2.Alanyl-tRNA Synthetase possesses a prototype structure 2 1.2.1.Aminoacylation domain of AlaRS for tRNAAla aminoacylation 2 1.2.2.Editing activity of AlaRS for removing misacylated tRNAAla 2 1.2.3.C-Ala domain of AlaRS constitutes the major tRNA-binding module 3 1.3.AlaRS specifically recognizes tRNAAla with G3:U70 3 1.4.The genome of Tupanvirus encodes a mini-AlaRS 4 1.5.Specific aims of this thesis 5 CHAPTER II METHODS 6 2.1.Cloning of AlaRS genes 6 2.2.Cloning of genes encoding tRNAAla and microhelixAla 6 2.3.Expression and purification of AlaRSs 7 2.4.Preparation of tRNAAla and microhelixAla 7 2.5.Aminoacylation assay 8 2.6.Electrophoresis mobility shift assay (EMSA) 8 2.7.Protein sequence alignment, phylogenetic analysis, and 3D structure prediction 9 CHAPTER III RESULTS 10 3.1.TuAlaRS comprises only aminoacylation domain 10 3.2.Tupanvirus genome encodes 4 isoacceptors of tRNAAla 10 3.3.Aminoacylation of TuAlaRS 11 3.4.tRNAAla Specificity of TuAlaRS 11 3.5.TuAlaRS efficiently aminoacylates microhelixAla 11 3.6.TuAlaRS robustly binds tRNAAla and microhelixAla 12 3.7.Kinetic parameters of tRNAAla and microhelixAla aminoacylation by TuAlaRS 13 3.8.P/T of TuAlaRS is not involved in recognition of tRNAAla 14 3.9.TuAlaRS mischarges Gly to tRNAAla to a higher level 14 3.10.Phylogenetic relationship of TuAlaRS with other AlaRSs 15 CHAPTER IV DISCUSSION 16 4.1.Mini-AlaRS specifically charges tRNAAla 16 4.2.TuAlaRS may evolve a new way to contact the acceptor stem of tRNAAla 17 4.3.TuAlaRS may have lost its C-Ala and editing domains lately during evolution 18 LIST OF FIGURES 20 LIST OF TABLES 32 APPENDIX I Plasmid List 33 APPENDIX II Primer & Synthesized Nucleotides 35 REFERENCES 37
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指導教授	王健家教授(Chien-Chia Wang)	審核日期	2022-7-18
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