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研究生:賴志昌
論文名稱:探討酵母菌Glycyl-tRNA合成酵素之生化活性
指導教授:王健家
學位類別:碩士
校院名稱:國立中央大學
系所名稱:生命科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:57
中文關鍵詞:酵母菌
外文關鍵詞:Glycyl-tRNA
相關次數:
  • 被引用被引用:0
  • 點閱點閱:262
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  • 下載下載:62
  • 收藏至我的研究室書目清單書目收藏:0
中文摘要
在酵母菌Saccharomyces cerevisiae 中蛋白質的合成同時在細胞
質及粒腺體內進行,因此這兩個地方皆需要有一套屬於自己的tRNA
合成酵素以供蛋白質合成之用,在細胞核內一般具有兩組不同的
tRNA 合成酵素基因,分別產生細胞質及粒腺體的酵素。雖然酵母菌
內也有兩個不同的GlyRS 基因(GRS1 及GRS2 ),但是其中一個(GRS2)
似乎為一假性基因(pseudogene),不具生理功能。本實驗主要目的是
要探求GlyRS1 和GlyRS2 之生化活性,並比較GlyRS1 及GlyRS2 在
演化上的關係。我們的結果顯示GlyRS2 處於低濃度時並不具有胺醯
化活性,在高濃度時才有活性;而GlyRS1 在低濃度時即具有很高的
胺醯化活性。GlyRS2 對大腸桿菌與酵母菌的tRNA 活性相差不多,
相對地,GlyRS1 則特別偏好酵母菌tRNA 。但在演化後期,GlyRS2 的
功能逐漸被GlyRS1 所取代,終究退化成一個假性基因。我們的結果
暗示在演化早期GlyRS1 及GlyRS2 可能分別負責細胞質及粒腺體的
tRNA 胺醯化作用。

英文摘要
In the yeast Saccharomyces cerevisiae, protein synthesis takes place
in two subcellular compartments: the cytosol and the mitochondria. Each
of these two compartments requires a complete set of tRNA synthetases
for function. Generally, these two sets of tRNA synthetases are encoded
by distinct nuclear genes. An recent report provided evidence that GRS1
encodes both the cytoplasmic and mitochondrial GlyRS activities, while
GRS2 is a pseudogene. The specific aims of this thesis are to explore the
biochemical activities of GlyRS1 and GlyRS2 and to further dissect the
evolutionary relationship between these two homologous genes.
Interestingly, although GRS2 is nonfuncuional in vivo, purified GlyRS2 ,
when used at high concentration, charges yeast tRNA at a significant rate.
Moreover, GlyRS2 cannot discriminate between yeast and E.coli tRNAs,
while GlyRS1 strongly prefers yeast tRNAs. Thus, our results provide an
evolutionary basis that ancient GRS1and GRS2 encode the cytoplasmic
and mitochondrial GlyRS activities, respectively.

iii
目錄
----------------------------------------------------------- 1
----------------------------------------------------------- 2
-------------------------------------------------------- 3
Ⅰ. Aminoacyl-tRNA synthetases (aaRSs )的簡介 -------------3
------------------------------------------------- 3
------------------------------------------------- 5
Ⅱ. 酵母菌(Saccharomyces cerevisiae) Glycyl-tRNA synthetase
(GlyRS )的簡介---------------------------------------------------- 7
Ⅲ. 研究目的 ----------------------------------------------------- 8
材料與方法 ---------------------------------------------- 10
---------------------------------------------- 10
Ⅱ.構築酵母菌的GlyRS1 及GlyRS2 融合蛋白質於酵母菌選殖載
體 ---------------------------------------------------------- 12
Ⅲ. GlyRS 的活體內分析:互補試驗(Complementation assay )iv
----------------------------------------------------------------- 17
Ⅳ. 用西方吸漬法測定GlyRS1 及GlyRS2 於酵母菌中的表現
--------------------------------------------------------------- 17
Ⅴ.純化GlyRS1 融合蛋白質------------------------------------- 19
Ⅵ.純化GlyRS2 融合蛋白質------------------------------------- 22
Ⅶ.GlyRS 的活體外分析:胺醯化作用分析(Aminoacylation assay )
--------------------------------------------------------------- 24
第三章 結果 --------------------------------------------------------- 26
Ⅰ.構築酵母菌的GlyRS1 及GlyRS2 融合蛋白質於酵母菌選殖
載體------------------------------------------------------------ 26
Ⅱ.GlyRS 的活體內分析:互補試驗(Complementation assay )
-----------------------------------------------------------------27
Ⅲ.純化GlyRS1 融合蛋白質-------------------------------------29
Ⅳ. 純化GlyRS2 融合蛋白質------------------------------------- 34
Ⅴ. 用西方吸漬法測定純化出之GlyRS1 及GlyRS2 分子量
----------------------------------------------------------------- 35
v
Ⅵ.GlyRS 的活體外分析:胺醯化作用分析(Aminoacylation
assay )------------------------------------------------------- 37
第四章 討論 --------------------------------------------------------- 42
第五章 參考文獻 --------------------------------------------------- 44
第六章 附錄 --------------------------------------------------------- 48
表目錄 ---------------------------------------------------------------- vi
圖目錄 ---------------------------------------------------------------- vii
vi
表目錄
表一. aaRS 的分類 -------------------------------------------------- 6
表二. Class Ⅰ及Class ⅡaaRS 的特徵 ------------------------------ 7
表三:酵母菌GlyRS 酵素之純化表---------------------------------35
表四:GlyRS1 及GlyRS2 分別對酵母菌及大腸桿菌tRNA 之催
化效率表------------------------------------------------------41
vii
圖目錄
圖一. 胺醯化作用 ----------------------------------------------------- 3
圖二. aaRS 在細胞中的功能及角色 ---------------------------------- 5
圖三. 構築GlyRS 在酵母菌選殖載體的圖示 ---------------------- 16
圖四. 酵母菌GRS1 及GRS2 基因之選殖---------------------------- 26
圖五. 酵母菌GlyRS 於5-FOA plate 上的生長---------------------- 28
圖六. 純化GlyRS1-His6 融合蛋白質之第一種純化系統DEAE(1)
-------------------------------------------------------------------29
圖七. 純化GlyRS1-His6 融合蛋白質之第二種純化系統DEAE(2)
------------------------------------------------------------------ 30
圖八.純化GlyRS1-His6 融合蛋白質之第三種純化系統
Hydroxylapatide ------------------------------------------------ 31
圖九.純化GlyRS1-His6 融合蛋白質之第四種純化系統 HiTrap-Q 管
柱---------------------------------------------------------------- 32
圖十.以SDS-PAGE 分析以不同純化系統純化出之GlyRS1-His6 融合
蛋白質的純度----------------------------------------------- 33
圖十一. 以SDS-PAGE 分析以不同純化系統純化出之GlyRS2-His6 融
合蛋白質的純度-------------------------------------------------- 34
圖十二. 西方吸漬法(Western blot)檢測並確認純化出之GlyRS1-His6
及GlyRS2-His6 融合蛋白質的分子量---------------------- 36
圖十三. GlyRS1 之胺醯化作用分析(Aminoacylation assay )-------- 37
圖十四. GlyRS1 對酵母菌及大腸桿菌tRNA 之胺醯化作用
(Aminoacylation )反應之比較------------------------------- 38
圖十五. GlyRS2 之胺醯化作用分析(Aminoacylation assay )--------39
圖十六. GlyRS2 對酵母菌及大腸桿菌tRNA 之胺醯化作用
(Aminoacylation )反應之比較------------------------------ 40

44
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