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姓名 葉曜榮(Yao-Jung Yeh)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 探討酵母菌Glutaminyl-tRNA synthetase 對於粒腺體功能之影響
(Is glutaminyl-tRNA synthetase essential for themitochondrial function of yeast?)
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摘要(中) Aminoacyl-tRNA (簡稱aa-tRNA) 的合成,對於蛋白質的生合成是非常重要的步驟,通常是藉由tRNA合成酶將胺基酸接到相對應的tRNA上。不過在Gln-tRNAGln的合成上卻是一個例外。在之前對於Saccharomyces cerevisiae的研究中指出,細胞質的glutaminyl-tRNA synthetase (簡稱GlnRS )是由GLN4基因所提供的,最近的研究發現細胞質的GlnRS會送到粒腺體中參與Gln-tRNAGln的合成。在本篇論文中我們利用細胞質的valyl-tRNA synthetase作為回報基因進一步找出其粒腺體標的訊號的範圍為包含第524 ~ 564號胺基酸,這段序列接近ATP的結合位 (KMSKS保留區域),這是少數的例子,其粒腺體標的訊號位於在內部序列當中同時也與主要的催化區重疊,但是對於細胞質的GlnRS是怎樣利用一段非傳統的N端粒腺體標的訊號去作用,其機制還不清楚。在Schizosaccharomyces pombe中並沒有發現有粒腺體標的訊號存在,所以我們認為這樣的標的訊號並不是所有酵母菌都擁有的。藉由功能性互補實驗、回報基因測試,發現GlnRS對於粒腺體功能的維持並不是必要的,因此在粒腺體中可能同時存在兩條途徑去合成Gln-tRNAGln。
摘要(英) Aminoacyl-tRNA (aa-tRNA) formation, an essential process in protein biosynthesis, is generally achieved by direct attachment of an amino acid to cognate tRNA by the aa-tRNA synthetases. An exception is Gln-tRNAGln synthesis. In Saccharomyces cerevisiae, the cytoplasmic glutaminyl-tRNA synthetase (GlnRS) activity is provided by the translational product of GLN4. Previous reports showed that this cytoplasmic GlnRS is also involved in mitochondrial Gln-tRNAGln synthesis. In this thesis, functional mapping using the cytoplasmic form of valyl-tRNA synthetase as the passenger protein identifies the peptide containing amino acids 524 ~ 564 of the enzyme as the mitochondrial targeting signal (MTS), which is close to the ATP-binding site (KMSKS conserved motif). This is one of the few examples, where MTS is embedded in the internal sequence, and is overlapped with the catalytic core domain of the enzyme. However, the detailed mechanism that enables GlnRS to be imported into mitochondria is not clear. In contrast, no MTS is found in Schizosaccharomyces pombe GlnRS, so this may be not a common feature for all yeast GlnRSs. Complementation tests further suggest that yeast GlnRS is not essential for mitochondrial function, and may serve as a redundant system for Gln-tRNAGln synthesis in mitochondria.
關鍵字(中) ★ tRNA 合成酶
★ 酵母菌
關鍵字(英) ★ GlnRS
★ Aminoacyl-tRNA synthetase
★ AARS
論文目次 中文摘要 i
英文摘要 ii
誌 謝 iii
目 錄 iv
圖 目 錄 vii
表 目 錄 viii
縮寫檢索表 ix
第一章 緒論 1
1.1 Aminoacyl-tRNA synthetases (aaRSs)的簡介 1
1.2原核與真核細胞在轉譯方式上的差異 3
1.3 Glutaminyl-tRNA synthetase (GlnRS)的簡介 4
1.4非專一性的tRNA結合蛋白 6
1.4.1 Arc1p 7
1.4.2 Ad(ScGlnRS) 7
1.5粒腺體標的訊號的特性 8
1.6研究目的 9
第二章 材料與方法 10
2.1菌株、載體及培養基 10
2.2大腸桿菌勝任細胞的製備與轉型作用 11
2.2.1大腸桿菌勝任細胞的製備 11
2.2.2大腸桿菌勝任細胞的轉型作用 (transformation) 12
2.3酵母菌勝任細胞的製備與轉型作用 12
2.3.1酵母菌勝任細胞的製備 12
2.3.2酵母菌勝任細胞的轉型作用 13
2.4質體之建構 14
2.5點突變 (Site-directed Mutagenesis) 14
2.6 GLN4剔除株的建構 16
2.7功能性互補試驗 (Complementation)―測試細胞質功能 17
2.8功能性互補試驗 (Complementation)―測試粒腺體功能 19
2.9蛋白質製備 20
2.10 SDS-PAGE之蛋白質分子量分析 21
2.11西方點墨法 (Western Blotting) 22
2.11酵母菌粒腺體的分離 (Enrichment of mitochondria) 24
第三章 結果 27
3.1利用回報基因找出ScGlnRS的粒線體標的訊號 27
3.2鑑定不同物種的GlnRS是否具有粒腺體標的訊號 29
3.3 ScGlnRS對於粒腺體功能不是必要的 31
3.4鑑定PgGlnRS是否同時具有細胞質及粒腺體的異構型 33
3.5利用西方式點墨法證實Arc1p在細胞中的分佈情形 34
第四章 討論 36
4.1 ScGlnRS對於粒腺體功能必要性之探討 36
4.2 Arc1p同時會在細胞質及粒腺體中分布 38
第五章 參考文獻 40
圖表 44
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桂妤 (2006)一個雙重功能的酵母菌tRNA合成酶之研究。中央大學碩士
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指導教授 王健家(Chien-Chia Wang) 審核日期 2008-7-1
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