Functional characterization of a noncanonical ProRS in Toxoplasma gondii

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伊凡緹(Indira Rizqita Ivanesthi) 查詢紙本館藏

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生命科學系

論文名稱

(Functional characterization of a noncanonical ProRS in Toxoplasma gondii)

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摘要(中)

Aminoacyl–tRNA synthetases (aaRSs) 是一群古老的酵素，它們的主要功能是
將一個胺基酸接到相對應的tRNA，接著aa-tRNA會被送去核醣體參與蛋白質合成。
在真核細胞內，蛋白質合成發生在細胞質及胞器(例如粒線體)，因此細胞會合成二
套aaRSs，其中一套作用在細胞質，另一套作用在粒腺體。一般而言，對應一個特
定胺基酸的細胞質及粒線體aaRSs是由二個不同的細胞核基因解碼，但是也有少數
例外。在本研究中我們發現弓形蟲(Toxoplasma gondii) 只有一個 prolyl-tRNA
synthetase (TgProRS)基因，但是有二個不同的tRNAPro (一個是E-type，另一個是Ptype)，
細胞核基因解碼的tRNAPro作用在細胞質及粒線體，它的序列屬於E-type；
頂質體基因解碼的tRNAPro作用在頂質體, 它的序列屬於P-type。序列比對及親緣演
化關係顯示TgProRS屬於 E-type ProRS，胺醯化活性測試也顯示這個酵素能有效地
胺醯化E-type tRNAPro，但是胺醯化 P-type tRNAPro的效率則明顯偏低。可是，互補
試驗顯示這個酵素能有效取代酵母菌粒線體ProRS (P-type ProRS)，這也顯示這個酵
素能胺醯化P-type tRNAPro到某一個程度。總而言之，我們的研究結果顯示TgProRS
是一個非典型的ProRS，能同時胺醯化P-type及E-type tRNAsPr

摘要(英)

Aminoacyl–tRNA synthetases (aaRSs) belong to a group of ancient enzymes that
plays a key role in protein synthesis by attaching a specific amino acid to its cognate tRNA.
In eukaryotes, protein synthesis occurs not only in the cytoplasm but also in organelles. Thus,
two distinct sets of aaRSs are required, one for the cytoplasm and the other for organelles.
In most cases, the cytoplasmic and organellar isoforms of a given aaRS are encoded by two
different nuclear genes, each recognizing its own tRNA isoacceptors. In this research we
found that Toxoplasma gondii contains only one nuclear prolyl-tRNA synthetase (TgProRS)
gene, but it contains two different tRNAPro isoacceptors, a nuclear-encoded E-type tRNAPro
(tRNAn
Pro), which functions in the cytoplasm and mitochondria, and an apicoplast-encoded
P-type tRNAPro (tRNAa
Pro), which functions in apicoplast. Sequence alignment and
phylogenetic analysisindicated that TgProRS possesses the unique C-terminal appended
domain that is unique to an E-type ProRS. Amininoacylation assays showed that TgProRS
can efficiently charge yeast tRNAPro, which represents an E-type tRNAPro, but it can hardly
charge E. coli tRNAPro and B. thuringiensis tRNAPro, both of which represent a P-type
tRNAPro. However, contrary to our expectations, TgProRS robustly rescued a yeast
mitochondrial ProRS knockout strain when it was overexpressed, suggesting that TgProRS
can charge the yeast mitochondrial P-type tRNAPro to a level sufficient to maintain normal
mitochondrial function. Our results suggest that TgProRS is a noncanonical ProRS that
charges both the P- and E-types of tRNAPro

關鍵字(中)

★ aminoacyl-tRNA synthetase
★ genetic code
★ protein synthesis
★ translation
★ tRNA

關鍵字(英)

★ aminoacyl-tRNA synthetase
★ genetic code
★ protein synthesis
★ translation
★ tRNA

論文目次

ABSTRACT (in Chinese)…………………………………………………………………………… i
ABSTRACT…………………………………………………………………………………….…iii
ACKNOWLEDGEMENT…………………………………………………………………………iv
TABLE OF CONTENT…………………………………………………………………………….v
LIST OF FIGURES……………………………………………………………………………….vii
ABBREVIATION………………………………………………………………………………..viii
CHAPTER I INTRODUCTION 1
1.1 Aminoacyl-tRNA synthetase 1
1.2 Prolyl-tRNA synthetase 1
1.3 The identity element tRNAPro and its recognition by ProRS 2
1.4 Toxoplasma gondii ProRS and tRNAsPro 3
1.5 Specific aim 4
CHAPTER II 5
MATERIALS and METHODS 5
2.1 Construction of yeast cytoplasmic ProRS knockout strain 5
2.2 Cloning of the genes encoding Toxoplasma gondii ProRS 5
2.3 Heterologous complementation assay for cytoplasmic ProRS activity 6
2.4 Heterologous complementation assay for mitochondrial ProRS activity 6
2.5 Western Blotting 7
2.6 Purification of TgProRS 7
2.7 In vitro Aminoacylation assay 8
2.8 In vitro transcription tRNA 8
CHAPTER III RESULTS 9
3.1 Sequence alignment analysis of TgProRS 9
3.2 TgProRS well expressed in E. coli and can be purified in homogeneity 9
3.3 TgProRS can charge yeast total tRNA in vitro 10
3.4 TgProRS barely charge E. coli total tRNA in vitro 11
3.5 TgProRS barely charge in vitro transcribed BttRNA in vitro 11
3.6 TgProRS can charge P-type tRNAPro in vivo 12
CHAPTER IV DISCUSSION 14
4.1 TgProRS prefer to charge E-type tRNAPro 14
4.2 TgProRS does not have conserved R residue to recognize P-type tRNAPro 15
4.3 Phylogenetic analysis of TgProRS 16
LIST OF FIGURES 18
REFERENCES 27
APPENDIX A 31
APPENDIX B 33

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指導教授

王健家(Chien-Chia Wang)

審核日期

2021-8-4

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