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研究生： 簡勤益
Chin-i Chien
論文名稱： Glycyl-tRNA synthetase 的演化與功能
Evolution and function of glycyl-tRNA synthetase
指導教授： 王健家
Chien-chia Wang
口試委員:
學位類別： 博士
Doctor
系所名稱： 生醫理工學院 - 生命科學系
Department of Life Science
論文出版年： 2015
畢業學年度： 103
語文別： 英文
論文頁數： 94
中文關鍵詞： 胺醯-tRNA合成酶 、跨物種援救 、鑑別基礎 、雙重功能的 、演化 、蛋白質合成 、轉譯作用
外文關鍵詞： aminoacyl-tRNA synthetase, cross-species rescue, discriminator base, dual functional, evolution, protein synthesis, translation
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    • Glycyl-tRNA synthetase (GlyRS) 的功能是將Glycine接到相對應的tRNAGly，這個酵素主要辦認tRNAGly上的discriminator base (N73)、anticodon (C35, C36) 和acceptor stem 的前三個鹼基配對 (1:72, 2:71 和3:70)。其中anticodon (C35, C36) 是最重要的辨認區位。自然界當中，GlyRS存在這兩種型式，一種是α2 type的二聚體型式，另一種則是α2β2 type的四聚體型式；原核及真核生物的tRNAGly上discriminator base (N73) 是不相同的，在原核生物N73是U，而在真核生物N73是A。酵母菌只有一個GlyRS基因，卻同時提供粒線體及細胞質功能，而且粒腺體及細胞質tRNAGly的N73都是A。第一部分，我們發現人類及阿拉伯芥之細胞質的GlyRS (α2 type) 可以取代酵母菌粒線體及細胞質的GlyRS功能 ，可是大腸桿菌的GlyRS (α2β2 type) 和阿拉伯芥胞器的 GlyRS [(αβ)2 type] 卻不能。然而將大腸桿菌GlyRS的α及β次單元融合，形成的αβ融合蛋白質可以取代酵母菌粒腺體的GlyRS功能，這種不同構型的GlyRS互相取代是一個非常罕見的現象。第二部分，線蟲的粒線體tRNAGly缺少T-arm，我們將酵母菌和線蟲的GlyRS (α2 type) 做胺基酸序列比對發現：線蟲GlyRS在胺基端多出一段胺基酸序列，我們想要知道這個額外的胺基酸序列是否與線蟲GlyRS辨認獨特的 tRNAGly有關，結果發現線蟲的細胞質及粒線體GlyRS都可以取代酵母菌GlyRS功能，然而刪除其WHEP domain 就喪失這個互補功能。胺醯化實驗顯示：純化的線蟲細胞質及粒線體GlyRS都能有效率地辨認線蟲細胞質tRNAGly，然而只有粒線體GlyRS可以辨認線蟲粒線體tRNAGly。我們的結果顯示：線蟲粒線體GlyRS胺基酸片段1-20是mitochondrial targeting signal，胺基酸片段21-64可能與粒線體tRNAGly辨認有關，胺基酸片段65-130可能是”非專一性tRNA結合區位”。

    Glycyl-tRNA synthetase (GlyRS) is an enzyme that specifically binds tRNAGly and catalyzes the synthesis of Gly-tRNAGly. The identity elements of tRNAGly for GlyRS include the discriminator base (N73), the last two nucleotides of the anticodon (C35, C36), and the acceptor stem region (1:72, 2:71 and 3:70). C35 and C36 are the most important elements for GlyRS recognition. Two oligomeric types of GlyRS are found in nature: a α2 type and a α2β2 type. The former has been identified in all three kingdoms of life and often pairs with tRNAGly that carries an A73 discriminator base, while the latter is found only in bacteria and chloroplasts and is almost always coupled with tRNAGly that contains U73. In the yeast Saccharomyces cerevisiae, a single GlyRS gene, GRS1, provides both the cytoplasmic and mitochondrial functions, and tRNAGly isoacceptors in both compartments possess A73. In the first part of this dissertation, we showed that Homo sapiens and Arabidopsis thaliana cytoplasmic GlyRSs (both α2-type enzymes) can rescue both the cytoplasmic and mitochondrial defects of a yeast grs1- strain, while Escherichia coli GlyRS (a α2β2-type enzyme) and A. thaliana organellar GlyRS [a (αβ)2-type enzyme] failed to rescue either defect of the yeast mull allele. However, a head-to-tail αβ fusion of E. coli GlyRS effectively supported the mitochondrial function. Our study suggests that a α2-type eukaryotic GlyRS may be functionally substituted with a α2β2-type bacterial cognate enzyme despite their remote evolutionary relationships. In the second part, we showed that mitochondrial tRNAGly of Caenorhabditis elegans lacks the entire T-arm. Sequence alignment showed that C. elegans GlyRS contains an N-terminal extension that is absent from its yeast counterpart. Our results showed that cytoplasmic and mitochondrial GlyRS isoforms effectively rescued a yeast GRS1 knockout strain. However, deletion of the N-terminal extension (WHEP domain) from the C. elegans GlyRS eliminated its cross-species rescue activity. Aminoacylation assays showed that both cytoplasmic and mitochondrial C. elegans GlyRS isoforms can charge the cytoplasmic tRNAGly isoacceptor. However, only the mitochondrial isoform can charge the mitochondrial tRNAGly isoacceptor. Our results suggested that the N-terminal peptide containing residues 1-20 is a mitochondrial targeting signal, peptide containing residues 21-64 is a specific mitochondrial tRNAGly recognition element, and peptide containing residues 65-130 is a nonspecific tRNA binding element.

    Table of Contents 中文摘要.i Abstract.ii Declaration.iii 誌謝.iv Tables of Contents.v List of Figures.vii List of Supporting Figures.viii List of Tables.ix Appendixes.x Abbreviations.xi Chapter I - Functional substitution of a eukaryotic glycyl-tRNA synthetase with an evolutionarily unrelated bacterial cognate enzyme.1 Abstract.2 Introduction.3 Materials and Methods.5 Results.8 Discussion.14 Charpter II - Analysis of the N-terminal appended domain of C. elegans glycyl-tRNA synthetase.18 Abstract.19 Introduction.20 Materials and Methods.21 Results.24 Discussion.31 References.35 List of Figures Figure 1. GlyRSs and tRNAsGly.39 Figure 2. Cross-species rescue assays for bacterial GlyRSs.40 Figure 3. Cross-species rescue assays for eukaryotic GlyRSs.42 Figure 4. Cellular distributions of human GlyRS.44 Figure 5. Aminoacylation activities of GlyRSs.45 Figure 6. Comparison of C. elegans GlyRS and various eukaryotic GlyRSs.47 Figure 7. Complementation assays for wild type and truncated form of C. elegans GlyRS.48 Figure 8. Comparison of yeast and C. elegans cytoplasmic and mitochondrial tRNAGly isoacceptors.50 Figure 9. Aminoacylation activities of wild type and truncated form of C. elegans GlyRS.51 Figure 10. Gel mobility-shift assay for the tRNA binding activities of the WHEP domain and cytosolic form and the WHEP-domain-deleted form C. elegans GlyRS.53 Figure 11. Converting EcGlnRS into a functional enzyme.55 Figure 12. Rescuing a defective C. elegans GlyRS mutant with Arc1p and Ad of yeast GlnRS.56 List of Supporting Figures Figure S1. Oligomeric structures of GlyRSs.46 List of Tables Table 1. Oligomeric stryctures of glycyl-tRNA synthetase and the discriminator base N73 of tRNAsGly.57 Appendixes Appendix 1. Yeast GRS1 homologues and their rescue activities.58 Appendix 2. Sequence comparison between GlyRS1 and GlyRS2.60 Appendix 3. Rescue activities of V. polyspora GRS2 and its derivatives.61 Appendix 4. Stress-inducible expression of V. polyspora GRS2.63 Appendix 5. Degradation of V. polyspora GlyRS1 and GlyRS2 in vivo.64 Appendix 6. Aminoacylation activities of V. polyspora GlyRS1 and GlyRS2 in vitro.65 Primer List.66 Plasmid List.70

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