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姓名 安媞卡(TITI RINDI ANTIKA)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 研究一個非典型alanyl-tRNA synthetase的作用機制
(Characterization of a noncanonical alanyl-tRNA synthetase)
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摘要(中) Aminoacyl-tRNA synthetase(aaRSs)是一群普遍存在的原始酵素,它可以將特定胺基酸連接至其相對應的tRNA,因此是蛋白質合成的關鍵酵素。由於蛋白質合成發生在真核細胞的細胞質和線粒體中,因此需要兩組不同的aaRSs,一組在細胞質中作用,另一組在線粒體中作用。在大多數情況下,細胞質和線粒體的aaRS同功異構酶是由兩個不同的細胞核基因編碼,每種aaRS藉由tRNA上的識別決定基來辨認其tRNA。 tRNAAla是一個非常特別的例子,它的唯一識別決定基是G3:U70,這種現象在細菌、真核細胞、古生菌都是一致的。 G3:U70由AlaRS中的兩個高度保留胺基酸Asp / Asn辨認。有趣的是,小鼠線粒體tRNAAla含有G1:U72,卻沒有G3:U70;而其AlaRS序列中也沒有這二個高度保留的胺基酸。我們想知道G1:U72是否真的是小鼠線粒體tRNAAla的識別決定基。我們選殖了小鼠線粒體AlaRS(AlaRSm或AARS2)的基因(AARS2),並定序AARS2及成熟的tRNAmAla。我們的研究結果顯示,小鼠粒線體AARS2無法胺醯化大腸桿菌的總和tRNA,但可以輕微胺醯化酵母菌的總和tRNA,進一步分析發現某些酵母菌非同源tRNA具有G1:U72,這些tRNA可能被AARS2錯誤胺醯化。然而類似含有G1:U72的tRNA並不存在大腸桿菌總和tRNA中。與該發現一致,當粒線體AARS2由一個超強的TEF1啟動子表達時,小鼠粒線體AARS2對酵母具有毒性。此外,我們的定序實驗顯示:小鼠tRNAmAla中的U5:U68不正常配對並沒有被轉錄後修正。此外,純化的小鼠粒線體AARS2可以胺醯化自己的tRNAmAla,但無法胺醯化人類的tRNAnAla,這表示AARS2的主要識別決定基不是G3:U70或反密碼,可能是G1:U72。我們的實驗結果證實小鼠粒線體AARS2是一個獨特的AlaRS,可以辨認不含G3:U70的tRNAAla。
摘要(英) Aminoacyl–tRNA synthetases (aaRSs) belong to a ubiquitous and ancient family of enzymes that play an important role in protein synthesis by attaching a specific amino acid to its cognate tRNA. Since protein synthesis takes place in both cytoplasm and mitochondria in eukaryotes, two distinct sets of aaRSs are required, one functioning in the cytoplasm and the other in mitochondria. In most cases, the cytoplasmic and mitochondrial isozymes of an aaRS are encoded by two different nuclear genes, each recognizing its own tRNA isoacceptor. The only identity element in tRNAAla is G3:U70 in the acceptor stem through all three kingdoms of life. G3:U70 is recognized by two highly conserved residues Asp/Asn in alanyl-tRNA synthetase (AlaRS). Interestingly, mouse mitochondrial tRNAAla contains G1:U72, instead of G3:U70. We were wondering whether G1:U72 actually serves as the identity element for mouse mitochondrial tRNAAla. Pursuant to this objective, we have cloned the gene encoding mouse mitochondrial AlaRS (AlaRSm or AARS2) and sequenced the mature tRNAmAla. Our results showed that mouse AARS2 can charge yeast unfractionated tRNAs to a significant level, suggesting that certain non-cognate tRNAs with G1:U72 might be mischarged by this enzyme. Consistent with the finding, mouse AARS2 was toxic to yeast when expressed from a vector with a strong TEF1 promoter, suggesting that AARS2 might cause mistranslation by attaching alanine to non-cognate tRNAs with G1:U72. In addition, our sequencing data confirmed that the U5:U68 mismatch in the acceptor stem of mouse tRNAmAla is retained during processing. Moreover, purified mouse AARS2 could charge its own tRNAmAla (with G1:U72), but failed to charge H. sapiens tRNAAla or E. coli total tRNAs, suggesting that the anticodon and G3:U70 are not the major identity elements for recognition of tRNAAla by mouse AARS2. Thus, mouse AARS2 is a unique AlaRS that can recognize tRNAAla without the canonical identity element G3:U70.
關鍵字(中) ★ 胺醯化
★ 識別決定基
★ 粒線體
★ 蛋白質合成
★ 轉譯
關鍵字(英) ★ aminoacylation
★ identity element
★ mitochondria
★ protein synthesis
★ tRNA
論文目次 ABSTRACT i
ABSTRACT (in Chinese) ii
ACKNOWLEDGEMENT iii
TABLE OF CONTENT iv
LIST OF FIGURES vi
ABBREVIATION vii
CHAPTER I INTRODUCTION 1
1.1 Protein synthesis in mitochondria 1
1.2 Aminoacyl-tRNA synthetase 1
1.3 Alanyl-tRNA synthetase and tRNAAla 2
1.4 The canonical identity element G3:U70 of tRNAAla and its recognition by AlaRS 2
1.5 Mouse mitochondrial tRNAAla and AlaRS 3
1.6 Specific aim 4
1.7 Hypothesis 4
CHAPTER II MATERIALS AND METHODS 5
2.1 Cloning of the genes encoding mouse mitochondrial AlaRS and tRNAAla 5
2.2 Site Directed Mutagenesis 5
2.3 Heterologous complementation assay for cytoplasmic AlaRS activity 6
2.4 Western Blotting 6
2.5 Complementation assay using temperature-sensitive E. coli AlaRS strain 6
2.6 Sequencing the gene encoding mouse mitochondrial AlaRS 7
2.7 Sequencing of mature mouse mitochondrial tRNAAla 7
2.8 Purification of mouse mitochondrial AlaRS 7
2.9 In vitro transcription of tRNAAla 8
2.10 In vitro Aminoacylation assay 8
CHAPTER III RESULTS 10
3.1 Mouse AlaRSm causes toxicity to yeast 10
3.2 An orthogonal pair of mouse AlaRSm and tRNAmAla can support the growth of
temperature-sensitive E. coli AlaRS strain 10
3.3 Sequencing of the gene encoding mouse mitochondrial AlaRS 11
3.4 U5:U68 in mouse mitochondrial tRNAAla is retained after processing 11
3.5 In vitro transcription of WT and mutant mouse mitochondrial tRNAsAla 12
3.6 Mouse mitochondrial AlaRS cannot charge a canonical tRNAAla 12
CHAPTER IV DISCUSSION 13
4.1 MmAlaRSm is a noncanonical AlaRS enzyme 13
4.2 MmtRNAmAla is a noncanonical tRNAAla 13
4.3 MmAlaRSm causes toxicity to yeast 14
4.4 MmAlaRSm can charge tRNAAla with G1:U72, but not tRNAAla with canonical
identity element G3:U70 15
4.5 G1:U72 is the identity element of MmtRNAmAla, but not the anticodon. 16
REFERENCES 18
APPENDIX 36

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指導教授 Prof. Wang Chien Chia 審核日期 2019-7-10
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