Bacillus thuringiensis contains two prolyl-tRNA synthetases of different origins

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盧曼里(Luqman Fikri Amrullah) 查詢紙本館藏

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論文名稱

(Bacillus thuringiensis contains two prolyl-tRNA synthetases of different origins)

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摘要(中)

Prolyl-tRNA synthetase (ProRS)是一群古老的酵素，負責將胺基酸連接到相對應的 tRNAPro 以進行蛋白質合成。 ProRS包含三個保守序列域（1-3）。根據蛋白質序列及結構，ProRS可分為兩種類型：真核/古細菌類型（E-type），其特點是含有一個C端延伸域（CTD）；原核類型（P-type），其特徵是在保守序列域 2 和 3 之間存在一個插入序列 (INS)。E-type ProRS 主要存在於古細菌、真核生物（細胞質和植物細胞器）、和一些細菌中，而 P-type ProRS 是存在於大多數細菌和真核生物的線粒體中。ProRS的親緣演化分析顯示，幾乎所有細菌都含有一個P-type ProRS和一個P-type tRNAPro。然而，蘇雲金芽孢桿菌(B. thuringiensis)包含兩種類型的 ProRS，P-type (BtProRS1) 和 E-type ProRS (BtProRS2) ，但是只含一個P-type tRNAPro。 BtProRS1 包含一個 INS 但缺少一個 CTD。相反地，BtProRS2 包含 CTD ，但缺少 INS。為了研究這兩種 ProRS 的 tRNA 特異性，我們使用酵母菌 ProS2 剔除株來進行互補測定，並使用純化的 BtProRS 和 tRNAsPro 做胺醯化測定。我們的結果顯示 BtProRS1 和 BtProRS2 都可以提供酵母菌線粒體所需的 ProRS活性，這表示這兩種酵素都可以將酵母線粒體的 P-type tRNAPro 胺醯化，達到足以維持正常線粒體功能的水平。不幸的是，我們純化的BtProRS1 和 BtProRS2 都都無法有效地胺醯化酵母菌 tRNAPro、大腸桿菌tRNAPro 或 BttRNAPro。進一步的親緣演化分析顯示，並非所有芽孢桿菌屬都含有兩種類型的 ProRS，我們將進一步討論這一發現的生物學意義。

摘要(英)

Prolyl-tRNA synthetase (ProRS) is an enzyme that activates proline and attaches it to tRNAPro for protein synthesis. ProRS belongs to class IIa aminoacyl-tRNA synthetase, containing three conserved sequence domains (motifs 1-3). Based on functional domains, ProRS itself is divided into two types: eukaryotic/archaea-like type (E-type), which is characterized by the presence of a C-terminal extension domain (CTD), and prokaryotic-like type (P-type), which is characterized by the presence of an insertion domain (INS) between motifs 2 and 3. E-type ProRSs are primarily found in archaea, eukaryotes (cytoplasm and plant organelle), and some bacteria, while P-type ProRSs are found in most bacteria and mitochondria of eukaryotes. Phylogenetic analysis of ProRS showed that almost all bacteria contain one P-type ProRS and one P-type tRNAPro. However, the bacterium Bacillus thuringiensis contains both types of ProRS, a P-type (BtProRS1) and an E-type ProRS (BtProRS2), and a P-type tRNAPro. BtProRS1 contains an INS but lacks a CTD. On the contrary, BtProRS2 contains a CTD but lacks an INS. To investigate the tRNA specificities of these two ProRS enzymes, a heterologous complementation assay using a yeast ProS2 knockout strain and an aminoacylation assay using purified BtProRSs and tRNAsPro as substrates were carried out. We demonstrated herein that both BtProRS1 and BtProRS2 could rescue the yeast mitochondrial ProRS KO strain, suggesting that both enzymes can charge the P-type yeast mitochondrial tRNAPro to a level sufficient to maintain normal mitochondrial function. Unfortunately, neither BtProRS1 nor BtProRS2 charged yeast total tRNA, E. coli total tRNA, or BttRNAPro. Further phylogenetic analysis revealed that not all Bacillus species contain both types of ProRS. The biological significance of this finding will be further discussed.

關鍵字(中)

★ tRNA合成酶
★ 芽孢桿菌
★ 水平基因轉移
★ 蛋白質合成
★ 轉譯
★ tRNA

關鍵字(英)

★ aminoacyl-tRNA synthetase
★ Bacillus
★ horizontal gene transfer
★ protein synthesis
★ translation
★ tRNA

論文目次

ABSTRACT i
ABSTRACT (in Chinese) ii
ACKNOWLEDGEMENT iii
TABLE OF CONTENT iv
LIST OF FIGURES vi
ABBREVIATION vii
CHAPTER 1. INTRODUCTION 1
1.1 Protein synthesis in prokaryotes 1
1.2 Aminoacyl-tRNA synthetase 1
1.3 Two types of Prolyl-tRNA synthetase 2
1.4 Different ProRSs types recognize different tRNAsPro 3
1.5 Bacillus thuringiensis carrying two types of ProRS and one types of tRNAPro 4
1.6 Specific aim 5
1.7 Hypothesis 5
CHAPTER 2. MATERIALS AND METHODS 6
2.1 Phylogenetic analysis of ProRSs 6
2.2 Plasmid construction of Bacillus thuringiensis ProRSs and tRNAPro genes 6
2.3 Heterologous complementation assay for mitochondrial ProRS activity 7
2.4 Purification of Bacillus thuringiensis ProRSs 7
2.5 In vitro transcription of tRNAPro 8
2.6 In vitro aminoacylation assay 8
2.7 Sequencing the gene encoding Bacillus thuringiensis ProRSs 9
CHAPTER 3. RESULTS 10
3.1 B. thuringiensis contains two distinct ProRSs and one P-type tRNAPro 10
3.2 Heterologous complementation assays for mitochondrial ProRS activity 11
3.3 Purification of B. thuringiensis ProRSs and tRNAsPro substrates 12
3.4 In vitro aminoacylation assays 13
CHAPTER 4. DISCUSSION 14
4.1 B. thuringiensis has two types of ProRSs and one P-type tRNAPro 14
4.2 Both P-type BtProRS1 and E-type BtProRS2 can recognize P-type tRNAPro, in vivo 15
4.3 Both BtProRSs are inactive in vitro 16
4.4 The E-type ProRS was acquired via horizontal gene transfer. Such a scenario occurred
only in some Bacillus species. 17
4.5 Both BtProRSs functions remain canonical 17
REFERENCES 19
APPENDIX A 41
APPENDIX B 42
APPENDIX C 43
APPENDIX D 45

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指導教授

王健家(Chien-Chia Wang)

審核日期

2021-7-16

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