探討酵母菌細胞質 glutamyl-tRNA synthetase 的粒腺體標的訊號

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姓名

陳念慈(Nien-tzu Chen) 查詢紙本館藏

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生命科學系

論文名稱

探討酵母菌細胞質 glutamyl-tRNA synthetase 的粒腺體標的訊號
(Identification of the mitochondrial targeting signal of yeast glutamyl-tRNA synthetase)

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摘要(中)

酵母菌Saccharomyces cerevisiae中cytoplasmic glutamyl-tRNA synthetase (cGluRS) 是由細胞核基因GUS1解碼而來。在細胞質中cGluRS會將Glu接到相對應的tRNAGlu形成Glu-tRNAGlu，但是最近的研究報告指出cGluRS也會被送到粒腺體中參與Gln-tRNAGln的形成。在粒腺體中cGluRS首先會將Glu接到tRNAGln形成Glu-tNRAGln，再藉由Glu-tNRAGln amidotransferase的轉胺作用形成正確的Gln-tRNAGln。先前，我們實驗室指出，在細胞質中與cGluRS結合的分子Arc1p有粒腺體標的訊號，因此cGluRS有可能是藉由Arc1p而進入粒腺體的，經由點突變的實驗我們發現cGluRS和Arc1p各自攜帶粒腺體標的訊號，不需靠彼此交互作用而進入粒腺體。cGluRS的粒腺體標的訊號位於其胺基端1-29胺基酸。相反地，在酵母菌Schizosaccharomyces pombe及Candida albicans的cGluRS中並沒有發現類似的粒腺體標的訊號。而在Pichia guilliermodii、Candida albicans、Schizosaccharomyces pombe這三株酵母菌的Arc1p也沒有粒腺體標的訊號。

摘要(英)

In Saccharomyces cerevisiae, the cytoplasmic glutamyl-tRNA synthetase (cGluRS) is encoded by GUS1. In cytoplasm, cGluRS attaches Glu to the cognate tRNAGlu to form Glu-tRNAGlu. In addition to the cytoplasmic activity, cGluRS can enter the mitochodria to synthesize Glu-tNRAGln (a misacylated product), which is then converted to correct Gln-tRNAGln by Glu-tNRAGln amidotransferase. A pervious study in our lab indicated that Arc1p, which binds cGluRS in cytoplasm, also has a mitochondrial targeting signal. Our results showed that cGluRS and Arc1p each carry a mitochondrial targeting signal and thus can enter the mitochondria by itself. Next, we used the cytoplasmic form of valyl-tRNA synthetase as the reporter gene to map the mitochondrial targeting signal of cGluRS. As it turned out, this signal was embedded in the N-terminal amino acids 1-29 of cGluRS. In contrast, no mitochondrial targeting signal was found in Schizosaccharomyces pombe or Candida albicans GluRS. Moreover, Arc1p from Pichia guilliermodii, Candida albicans, and Schizosaccharomyces pombe does not possess a mitochondrial targeting signal.

關鍵字(中)

★ 粒腺體標的訊號

關鍵字(英)

★ GluRS
★ MTS

論文目次

中文摘要i
英文摘要ii
誌謝iii
目錄iv
圖目錄vii
縮寫檢索表viii
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2
1.3 Glutaminyl-tRNA synthetase (GluRS)的簡介4
1.4 非專一性的tRNA 結合蛋白5
1.4.1 Arc1p 5
1.4.2 Ad(ScGluRS)5
1.5 粒腺體標的訊號的特性6
1.6 研究目的6
第二章材料與方法8
2.1 菌株、載體及培養基8
2.2 大腸桿菌勝任細胞的製備與轉型作用 9
2.2.1 大腸桿菌勝任細胞的製備9
2.2.2 大腸桿菌勝任細胞的轉型作用 (transformation)10
2.3 酵母菌勝任細胞的製備與轉型作用10
2.3.1 酵母菌勝任細胞的製備10
2.3.2 酵母菌勝任細胞的轉型作用11
2.4 質體之建構 11
2.5 點突變 (Site-directed Mutagenesis)12
2.6 功能性互補試驗 (Complementation)―測試細胞質功能13
2.7 功能性互補試驗 (Complementation)―測試粒腺體功能14
2.8 蛋白質製備15
2.9 SDS-PAGE 之蛋白質分子量分析16
2.10 西方點墨法 (Western Blotting) 16
2.11酵母菌雙雜交系統 (Yeast Two-hybrid Assay)17
2.12 In Vitro Pull Down Assay 18
2.13酵母菌融合蛋白質的表現與純化18
第三章結果 22
3.1 測定Arc1p的粒腺體標的訊號22
3.2利用點突變方法證實Arc1p及ScGluRS的胺基端粒腺體標的訊號之活性23
3.3 利用two-hybrid及pull-down的方法證實Arc1p及ScGluRS胺基端的交互作用24
3.4利用回報基因找出ScGluRS的粒腺體標的訊號25
3.5 鑑定不同物種的GluRS是否具有粒腺體標的訊號25
3.6 鑑定不同物種的Arc1p是否具有粒腺體標的訊號26
第四章討論 28
4.1 ScGluRS 對於粒腺體功能必要性之探討28
4.2 ScGluRS的粒腺體標的訊號之預測30
第五章參考文獻 32

參考文獻

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葉曜榮 (2008)探討酵母菌Glutaminyl-tRNA synthetase對於粒腺體功能之影響。中央大學碩士論文 84頁

指導教授

王健家(Chien-chia Wang)

審核日期

2010-6-25

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