探討酵母菌Valyl-tRNA synthetase的生化活性

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姓名

林于慧(Yu-Hui Lin) 查詢紙本館藏

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生命科學系

論文名稱

探討酵母菌Valyl-tRNA synthetase的生化活性
(Exploring the biological fumction of yeast Valyl-tRNA synthetase)

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摘要(中)

不同於大部分的tRNA合成酵素，酵母菌中的細胞質與粒腺體Valine-tRNA合成酵素（ValRS）是由同一個細胞核基因所解碼出來;這兩個異構型唯一的不同處是在於新合成的粒腺體合成酵素多了一段由46個胺基酸組成的"粒腺體標的訊號"。雖然在活體外這兩個合成酵素有幾乎相同的活性，但是在活體內這兩個合成酵素因為存在不同的胞器內，所以無法互相取代。我們發現此合成酵素的胺基端具有一段富含Lysine的附加區段（胺基酸1~97），但是此附加區段並不直接參與胺醯化作用，也不是細胞內蛋白質合成功能之所需;利用two-hybrid系統我們發現ValRS的附加區段能夠活化細胞核內回報基因的轉錄作用，而這種轉錄活化功能並未發現於其他已知的aaRS附加區段，且其序列亦與已知的轉錄活化蛋白質不同，因此推斷可能是一個新的轉錄活化因子。

摘要(英)

In yeast, there are typically two distinct nuclear-encoded tRNA synthetases for each amino acids; one function in the cytoplasm and the other in the mitochondria. Previous studies showed that VAS1 is the only gene coding for valyl-tRNA synthetase （ValRS） in the yeast genome. This gene encodes both the cytoplasmic and mitochondrial forms of valyl-tRNA synthetase. These two isoforms have essentially identical polypeptide sequences except for a 46-amino acid leader at the N-terminus of the mitochondrial precursor, which functions as a mitochondrial targeting signal. In vitro, the two isoforms of ValRS cannot distinguish between tRNA species from the cytoplasm and mitochondria, but the enzymes cannot substitute for each other in vivo due to differential partitioning. Also, like many other eukaryotic tRNA synthetase, the cytoplasmic form of yeast ValRS has a lysine-rich polypeptide extension of 97 amino acids appended to its N-terminus. I found that the appended domain did not participate in aminoacylation and was largely dispensible for function in vivo. Surprisingly, our results showed that this appended domain exhibited a strong transcriptional activation activity that was never found in any other tRNA synthetase tested. This suggests that this appended domain may be a novel transcriptional activator.

關鍵字(中)

★ 酵母菌

關鍵字(英)

★ yeast
★ Valyl-tRNA synthetase

論文目次

第一章緒論
I. 何謂aaRS（Aminoacyl-tRNA synthetase;tRNA合成酵素） -------------------------------------------------------------------------1
A. aaRS的生化功能----------------------------------------------- 1
B. ClassI和ClassII aaRS----------------------------------------- 2
II. Prokaryote及Eukaryote中的aaRS ----------------------------3
A. 分佈情形---------------------------------------------------------3
B. aaRS形成complex -------------------------------------------- 3
III. aaRS除了胺醯化作用之外具有非傳統的功能-------------4
IV. 酵母菌中的aaRS-------------------------------------------------5
A. aaRS 的合成--------------------------------------------------5
B. 選擇性轉錄及轉譯作用 ------------------------------------5
C. Valyl-tRNA synthetase（ValRS）----------------------- 7
1. ValRS的生化特性---------------------------------------7
2. 酵母菌中的ValRS---------------------------------------7
第二章材料與方法------------------------------------------------ 10
I. 實驗材料-----------------------------------------------------------10
II. 實驗方法----------------------------------------------------------- 11
A. 核酸的製備---------------------------------------------------11
1. 質體DNA的小量純化---------------------------------11
2. 啟動子DNA的製備------------------------------------ 12
B. 大腸桿菌勝任細胞的製備與轉型作用------------------13
1. 大腸桿菌勝任細胞的製備-----------------------------13
2. 大腸桿菌勝任細胞的轉型作用-----------------------14
C. 酵母菌勝任細胞的製備與轉型作用---------------------15
1. 酵母菌勝任細胞的製備--------------------------------15
2. 酵母菌勝任細胞的轉型作用--------------------------15
D. 大腸桿菌融合蛋白質的表現、純化與濃縮------------16
1. 大腸桿菌融合蛋白質的表現--------------------------16
2. 大腸桿菌融合蛋白質的純化--------------------------17
3. 大腸桿菌融合蛋白質的濃縮--------------------------19
E. 酵母菌融合蛋白質的表現、純化與濃縮----------------20
F. 西方墨點法（Western blot）------------------------------20
1. 酵母菌粗萃物的製備 ----------------------------------20
2. 正十二烷硫酸鈉-聚丙醯胺凝膠電泳（Sodium dodecyl sulfate polyacrylamide gel electrophoresis，SDS-PAGE）------------------------21
3. 蛋白質的轉漬--------------------------------------------21
G. 胺醯化作用分析---------------------------------------------22
1. 蛋白質的濃度測定與稀釋-----------------------------22
2. 胺醯化作用-----------------------------------------------22
H. 聚丙烯醯胺親和力共電泳（Polyacrylamide Affinity Coelectrophoresis，PACE）製膠與電泳--------------24
第三章結果---------------------------------------------------------------25
I. 附加區段對於ValRS胺醯化的影響---------------------------25
II. ValRS是否具有調控自己基因表現的能力？---------------27
1. 酵母菌ValRS的附加區段不會鍵結DNA-----------29
2. 酵母菌ValRS不會鍵結DNA，也不具有專一性---30
第四章討論---------------------------------------------------------------32
參考文獻--------------------------------------------------------------------36
表------------------------------------------------------------------------------39
圖------------------------------------------------------------------------------42

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謝佳容，酵母菌Valyl-tRNA synthetase 附加區段的生物功能之探討，民國91年，國立中央大學生命科學研究所

指導教授

王健家(Chien-Chia Wang)

審核日期

2003-7-18

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