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論文名稱 真核細胞Thg1添加酵素的功能鑑定
(Functional characterization of eukaryotic Thg1 enzymes)
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摘要(中) 胺基酸-tRNA合成酶(aaRSs)是必需的轉譯酵素,其中每一種都催化特定胺基酸與其同源tRNA的結合。另外5′ G-1的存在是tRNAHis在所有三個生命領域中的獨特特徵。這種不尋常的G-1是tRNAHis的主要辨識決定基,對於胺基酸-tRNA合成酶(HisRS)的辨識至關重要。G-1可由兩種完全不同的機制產生:在細菌(和許多古細菌)中,G-1是由tRNAHis基因轉錄而來;在真核生物中,G-1是在tRNAHis轉錄後,由 Thg1酵素’’添加上去的,Thg1主要辨認tRNAHis的反密碼。一般而言,具有A73的tRNAHis是用轉錄後修飾的方法獲得G-1,具有C73的tRNAHis則是由基因轉錄直接而來。我們的結果顯示:人(Homo sapiens)、果蠅(Drosophila melanogaster)、和家蠶(Bombyx mori)的Thg1酵素都具有線粒體標的訊號(MTS),因此這些Thg1可以同時在細胞質及線粒體中作用。此外,我們發現這些Thg1可以同時將G-1添加在細胞質tRNAnHis (含有A73)和線粒體tRNAmHis (含有C73)。有趣的是,人類和果蠅Thg1酵素利用ATP依賴性機制將G-1加到tRNAnHis,卻是利用GTP依賴性機制將G-1加到tRNAmHis。本研究為真核細胞Thg1酵素如何作用在細胞質及線粒體提供了新的見解。
摘要(英) Aminoacyl-tRNA synthetases (aaRSs) are essential translation enzymes, each catalyzes the coupling of a specific amino acid to its cognate tRNAs. The presence of an additional 5’ guanosine residue (G-1) is a unique feature of tRNAHis in all three domains of life. This unusual G-1 residue is the major identity element of tRNAHis and is essential for recognition by histidyl-tRNA synthetase (HisRS). The functional significance of G-1 is evidenced by the existence of two entirely distinct mechanisms: G-1 is encoded in the tRNAHis gene of bacteria (and many archaea) and G-1 is added post-transcriptionally by tRNAHis guanylyltransferase (Thg1) in eukaryotes. Thg1 possesses an efficient 3’–5’polymerase activity that specifically adds the G-1 residue by recognizing the anticodon of tRNAHis. Typically, G-1 addition to tRNAHis with A73 is mediated via an ATP-dependent mechanism in eukaryotes, while G-1 addition to tRNAHis with C73 is mediated via a GTP-dependent mechanism in prokaryotes. We reported herein that like human Thg1, Thg1 homologs of Drosophila melanogaster and Bombyx mori also possess a mitochondrial targeting signal (MTS). As a result, these Thg1 enzymes can be targeted to both cytoplasm and mitochondria. Moreover, we found that these Thg1 enzymes can attach G-1 to both cytoplasmic tRNAsHis with A73 and mitochondrial tRNAsHis with C73. Interestingly, Drosophila Thg1 enzyme attaches G-1 to tRNAnHis via an ATP-dependent mechanism, while it attaches G-1 to tRNAmHis via a GTP-dependent mechanism. This study provides new insight into the mechanisms of tRNAHis maturation in the cytoplasm and mitochondria.
關鍵字(中) ★ tRNAHis guanylyltransferase
★ G-1
★ post-transcriptional modification
★ protein synthesis
★ translation
關鍵字(英) ★ tRNAHis guanylyltransferase
★ G-1
★ post-transcriptional modification
★ protein synthesis
★ translation
論文目次 ABSTRACT i
中文摘要 ii
ACKNOWLEDGMENTS iii
TABLE OF CONTENT iv
LIST OF FIGURES vi
ABBREVIATION viii
INTRODUCTION 1
I. Aminoacyl-tRNA synthetase (aaRSs): a group of essential translation enzymes. 1
II. Nearly all histidine tRNAs possess a universal extra guanine (G-1) at the 5’end. 1
III. G-1 plays essential roles in cell. 2
IV. The essential role of tRNAHis guanylyltransferase (Thg1) for cell viability. 2
MATERIALS AND METHODS 4
I. Construction and purification of Thg1 enzymes 4
II. Construction and in vitro transcription of tRNAsHis 5
III. G-1 addition assay 5
IV. Western Blot 5
V. Construction and purification of ScGAPDH 6
VI. Pull-down assays 6
VII. Yeast two-hybrid assay 7
VIII. Determination of G-1 status of Drosophila melanogaster tRNAmHis 7
CHAPTER I 9
I. Introduction 9
II. Results 10
1. The unspecific protein having a high affinity with ScThg1 in yeast cells was GAPDH 10
2. GAPDH did not directly associate with Thg1. 10
3. The interaction between GAPDH and Thg1 did not occur in the yeast nucleus. 10
4. GAPDH failed to interact with Thg1 in the yeast cytoplasm. 11
5. The efficiency of G-1 addition reaction was not assisted by GAPDH. 11
III. Discussion 13
CHAPTER II 15
I. Introduction 15
II. Results 17
1. Drosophila mitochondrial tRNAHis acquires G-1 through post-transcriptional processing. 17
2. Drosophila Thg1 catalyzes G-1 addition to both cytoplasmic and mitochondrial tRNAHis. 17
3. Human, Drosophila, and Bombyx Thg1 enzymes attach G-1 to both cytoplasmic tRNAHis (with A73) and mitochondrial tRNAHis (with C73). 17
4. Human Thg1 and Drosophila acts on tRNAnHis and tRNAmHis through two divergent mechanisms. 18
5. A proposed evolutionary relationships among members of the Thg1 family. 19
6. The highly conserved SDEYSF and HINNLYN motif in a eukaryote-type Thg1. 20
III. Discussion 21
REFERENCES 24
APPENDIX A 45
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指導教授 王建家(Chien-Chia Wang) 審核日期 2019-7-16
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