酵母菌valyl-tRNA synthetase附加區段的
生物功能之探討

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謝佳容(Chia-Jung Hsieh) 查詢紙本館藏

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生命科學系

論文名稱

酵母菌valyl-tRNA synthetase附加區段的生物功能之探討
(Elucidating the biological functions of the appended domain of yeast ValRS )

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摘要(中)

在酵母菌的系統中，相對應於每種胺基酸分別有兩套解碼自細胞
核基因的tRNA 合成酵素。其中一套用於細胞質中的蛋白質合成作
用，另一套則是在粒線體中執行反應。然而，在之前的研究中顯示
VAS1 是酵母菌染色體中唯一能解碼valine tRNA 合成酵素的基因。此
基因利用選擇性轉錄及轉譯作用同時合成細胞質及粒線體的valine
tRNA 合成酵素。這兩個同功酵素具有幾乎完全相同的胺基酸序列，
但是在粒線體valine tRNA 合成酵素的胺基端多了一組由46 個胺基酸
組成的”粒線體標的訊號”，由於這兩個tRNA 合成酵素存在於不同的
胞器內，使得它們在功能上並不能相互的取代。有趣的是，相較於原
核生物的valine tRNA 合成酵素，酵母菌的細胞質valine tRNA 合成
酵素在其胺基端多了一段由98 個胺基酸組成的附加區段。序列分析
的結果顯示這個區段含有許多帶正電荷的胺基酸，不同於大部分合成
酵素的附加區段，純化的valine tRNA 合成酵素的附加區段並不會鍵
結tRNA ，在功能上也是可有可無，將大部分的附加區段由合成酵素
中刪除並不影響它的活體內及活體外功能。值得注意的是，我們發現
這個附加區段本身具有活化轉錄的功能，或許valine tRNA 合成酵素
本身就是一個轉錄活化因子。

摘要(英)

In yeast, there typically are two distinct nuclear-encoded tRNA
synthetases for each amino acid; one functions in the cytoplasm and the
other in the mitochondria. However, evidence shows that VAS1 is the only
gene coding for valyl-tRNA synthetase (ValRS) in the complete yeast
genome. This gene encodes not only the cytoplasmic form of ValRS but
also its mitochondrial isoform. These two ValRS enzymes have
essentially identical polypeptide sequences, except for a 46-amino acid
leader peptide at the N-terminus of the mitochondrial precursor, which
functions as a mitochondrial targeting signal. Biochemical studies show
that these two ValRS isoforms cannot distinguish tRNA species from the
cytoplasm and mitochondria in vitro. Despite these similarities, these two
ValRS species cannot substitute for each other in vivo, presumably due to
differential partitioning. We show here that the appended domain of yeast
ValRS, which is absent in its prokaryotic counterparts, is largely
dispensable for the enzyme’s aminoacylation as well as complementing
activities. Moreover, unlike the appended domains of other yeast tRNA
synthetases, the appended domain of yeast ValRS does not appear to bind
tRNA. Interestingly, this dispensable domain exhibits a strong
transcriptional activation activity in a two-hybrid test, suggesting that
ValRS might be involved in regulation of gene expression.

關鍵字(中)

★ tRNA合成酶
★ 胺醯化作用

關鍵字(英)

★ valine
★ valine tRNA synthetase
★ Aminoacylation

論文目次

目錄
目錄-----------------------------------------------------------------------I
『表』目錄----------------------------------------------------------------------IV
『圖』目錄-----------------------------------------------------------------------V
縮字表--------------------------------------------------------------------------VII
第一章緒論--------------------------------------------------------------------1
壹、簡介tRNA 合成酵素--------------------------------------------1
一、tRNA 合成酵素的生化功能
二、tRNA 合成酵素的分類
三、高等真核生物中tRNA 合成酵素的特性
貳、酵母菌中tRNA 合成酵素的分佈-----------------------------3
一、蛋白質在哪裡合成？
二、要如何由一個基因得到兩個蛋白質產物？
參、簡介Valyl-tRNA synthetase-----------------------------------5
一、 ValRS 的生化特性
二、酵母菌中的ValRS
第二章材料與方法----------------------------------------------------------7
壹、實驗材料-----------------------------------------------------------7
一、大腸桿菌株
二、酵母菌株
三、載體
四、核酸引子
貳、實驗方法-----------------------------------------------------------8
一、核酸的製備
二、大腸桿菌勝任細胞的製備與轉型作用
三、酵母菌ValRS 附加區段基因的增幅與選殖
四、酵母菌勝任細胞的製備與轉型作用
五、大腸桿菌融合蛋白質的表現與純化
六、酵母菌融合蛋白質的表現與純化
七、西方墨點法
八、酵母菌互補作用試驗
九、胺醯化作用分析
十、聚丙烯醯胺親和力共電泳
第三章結果------------------------------------------------------------------24
壹、酵母菌ValRS 的附加區段具有轉錄活化作用------------24
貳、酵母菌ValRS 的附加區段對於ValRS 缺失株的互補能
力並不重要-----------------------------------------------------25
參、酵母菌ValRS 的附加區段並不參與ValRS 的胺醯化作
用-----------------------------------------------------------------26
肆、酵母菌ValRS 的附加區段不會鍵結tRNA----------------27
伍、酵母菌ValRS 的附加區段不會鍵結DNA-----------------28
討論------------------------------------------------------------------30
參考文獻------------------------------------------------------------------------32
表----------------------------------------------------------------------------------38
圖----------------------------------------------------------------------------------40
附錄-------------------------------------------------------------------------------58

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指導教授

王健家(Chen-Chia Wang)

審核日期

2002-7-18

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