酵母菌粒線體Gln-tRNAGln的形成

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姓名

廖芝淇(Chih-chi Liao) 查詢紙本館藏

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生命科學系

論文名稱

酵母菌粒線體Gln-tRNAGln的形成
(Formation of Gln-tRNAGln in the yeast mitochondria)

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摘要(中)

在細胞中，有兩種合成Gln-tRNAGln的路徑，一種是和GlnRS有關的直接路徑，另一種是和轉胺酶GatFAB有關的間接路徑。之前的研究發現，在酵母菌Saccharomyces cerevisiae中，部分細胞質的GlnRS會被送到粒線體中，因此他們推測酵母菌粒線體可能是利用直接路徑來合成Gln-tRNAGln，但是最近的研究發現酵母菌粒線體是經由間接路徑來合成Gln-tRNAGln，那麼酵母菌粒線體中GlnRS執行甚麼功能有待進一步的研究。在本篇論文中，我們利用報導基因試驗以及免疫螢光分析來標定酵母菌GlnRS的粒線體標的訊號，實驗結果顯示: 粒線體標的訊號不在其胺基端，而是位於GlnRS中靠近活化區的一段序列。這是一個非常特別的例子，GlnRS的標的訊號在蛋白質進到粒線體後不會被切除。另外，我們發現，在EcGlnRS的胺基端接上Arc1p可以提供GLN4 剔除株生長所必須的酵素活性，若進一步在此融合蛋白質的胺基端加上一段粒線體標的訊號則此融合蛋白質可以取代粒線體內間接合成Gln-tRNAGln的路徑，這些發現突顯了基因平行轉移的可能性，利用直接合成Gln-tRNAGln的路徑取代間接合成路徑。

摘要(英)

There are two pathways for Gln-tRNAGln formation in the cell, a direct pathway, which involves GlnRS, and an indirect pathway, which involves a GatFAB transamidase. In Saccharomyces cerevisiae, early research indicated that a portion of cytoplasmic GlnRS was transported into the mitochondria. It was thus proposed that the yeast mitochondria may employ a direct pathway to form Gln-tRNAGln. However, a recent study argued that an indirect pathway is actually involved in the synthesis of mitochondrial Gln-tRNAGln. As a result, the true biological function of the imported GlnRS in the mitochondria is still elusive. In this study, we used reporter gene assays, and immunofluorescence analysis to map the mitochondrial targeting signal of yeast GlnRS. Our results showed that the signal is located at an internal segment close to the active site of the enzyme. This might be one of few examples of mitochondrial matrix proteins that use an uncleavable internal sequence as the mitochondrial targeting signal. On the other hand, we found that fusion of Arc1p to EcGlnRS enables the bacterial enzyme to rescue the growth defect of a GLN4 knockout strain. Further fusion of a mitochondrial targeting signal to the fusion enzyme Arc1p-EcGlnRS enabled the enzyme to replace the indirect pathway for Gln-tRNAGln formation in the mitochondria. These findings underscore the possibility of a horizontal transfer event, where an indirect pathway for Gln-tRNAGln formation is substituted for by a direct pathway.

關鍵字(中)

★ tRNA合成酶
★ 酵母菌
★ 粒線體

關鍵字(英)

★ tRNA synthetase
★ yeast
★ mitochondria

論文目次

中文摘要 i
ABSTRACT ii
誌謝 iii
目錄 iv
圖目錄 vii
表目錄 ix
縮寫檢索表 x
第一章緒論 - 1 -
1.1 Aminoacyl-tRNA synthetases (aaRSs)的簡介 - 1 -
1.1.1 aaRS的功能 - 1 -
1.1.2 aaRS 的分類 - 2 -
1.2 Gln-tRNAGln 的合成 - 3 -
1.2.1合成Gln-tRNAGln 的途徑 - 3 -
1.2.2 間接合成Gln-tRNAGln - 4 -
1.2.3 酵母菌Saccharomyces cerevisiae中Gln-tRNAGln的合成 - 4 -
1.3 Glutaminayl-tRNA synthetase (GlnRS)的簡介 - 5 -
1.3.1 GlnRS的生化特性 - 5 -
1.3.2 GlnRS的演化 - 6 -
1.4 粒線體標的訊號 ( Mitochondrial targeting signal) - 7 -
1.5 非專一性tRNA結合蛋白 - 8 -
1.5.1 Arc1p - 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 - 12 -
2.2.2大腸桿菌勝任細胞的轉型作用 (transformation) - 13 -
2.3 酵母菌勝任細胞的製備與轉型作用 - 13 -
2.3.1酵母菌勝任細胞的製備 - 13 -
2.3.2酵母菌勝任細胞的轉型作用 - 14 -
2.4 質體之選殖 - 14 -
2.5 點突變 (Site-directed Mutagenesis) - 15 -
2.6 功能性互補試驗 (Complementation)―測試細胞質功能 - 16 -
2.7 功能性互補試驗 (Complementation)―測試粒腺體功能 - 17 -
2.8 蛋白質製備 (Protein preparation) - 19 -
2.9 SDS-PAGE之蛋白質分子量分析 - 20 -
2.10 西方點墨法 (Western Blotting) - 20 -
2.11 粒線體標的訊號報導基因試驗 (MTS reporter gene assay) - 22 -
2.12 免疫螢光分析 (immunofluorescence analysis) - 23 -
第三章結果 - 24 -
3.1 ScGlnRS粒線體標的訊號位於胺基酸524-564區間。 - 24 -
3.2 點突變讓ScGlnRS胺基酸片段失去粒線體標的訊號的活性。 - 24 -
3.3 特定的點突變使全長的ScGlnRS無法進入粒線體。 - 26 -
3.4 真核生物間GlnRSs的相互關係。 - 28 -
3.5 原核和真核生物GlnRSs之間的關係。 - 31 -
第四章討論 - 35 -
4.1 ScGlnRS的粒線體標的訊號 - 35 -
4.2 GlnRS的演化 - 37 -
參考文獻 - 40 -
圖表 - 44 -

參考文獻

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指導教授

王健家(Chien-chia Wang)

審核日期

2011-6-26

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