探討一個真核tRNA合成酶的附加區段之轉錄活化活性

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姓名

林明琁(Grace Lin) 查詢紙本館藏

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生命科學系

論文名稱

探討一個真核tRNA合成酶的附加區段之轉錄活化活性
(A transcriptional activation domain in a eukaryotic tRNA synthetase)

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摘要(中)

之前的研究顯示，酵母菌的valyl-tRNA 合成酶在胺基端有一段由
135 個胺基酸所組成並富含離胺酸（lysine）的多肽序列，這是原核細
胞的同源酵素所缺乏的，因此稱為附加區段。本實驗室之前的實驗結
果發現附加區段雖然帶有許多正電荷卻對酵母菌的tRNA沒有特別的
親和力，且刪除附加區段的酵素在in vitro 的功能和野生型的ValRS
又差不多。而進一步想利用yeast two-hybrid 的方式去篩選會和附加
區段作用的蛋白質，卻意外的發現附加區段自己本身就是一個轉錄活
化區段（transcriptional activation domain）。因此本論文會針對ValRS
附加區段對酵素活性的影響以及附加區段的轉錄活化特性去做進一
步的探討。首先用刪除株互補能力測試發現附加區段對ValRS 在in
vivo 中的酵素活性沒有明顯的影響，而初步的結果也顯示附加區段可
將蛋白質送到細胞核。而經片段刪除後也確認提供轉錄活化特性的最
小序列片段包含第98 到135 胺基酸。較令人意外的是：酵母菌其他
的aaRS 雖然也有一個富含離胺酸的附加區段，但是並沒有轉錄活化
的特性，而相近物種之ValRS 的附加區段也缺乏此功能。此例子是第
一次在真核細胞的aaRS 中發現有轉錄活化特性的附加區段，而探討
其在細胞內的生化意義之研究目前仍在持續進行中。

摘要(英)

Previous studies show that valyl-tRNA synthetase of Saccharomyces cerevisiae
contains an amino-terminal, 135-amino acid lysine-rich polypeptide extension which
is absent from its bacterial relatives. We show here that this polypeptide extension is
largely dispensable for the aminoacylation activity of the enzyme in vivo and in vitro.
A truncated version of the enzyme with up to 83 amino-terminal residues deleted
rescues the growth defect of a yeast strain deficient in valyl-tRNA synthetase activity,
while an enzyme missing the lysine rich cluster retains much of its aminoacylation
activity in vitro. Furthermore, although rich in positively charged residues, this
polypeptide extension binds poorly to yeast tRNAs in vitro. These results suggest that
the appended domain of the translational enzyme may not be involved in tRNA
aminoacylation. Remarkably, when the appended domain is fused to a DNA-binding
protein (LexA), the resultant fusion can effectively activate the transcription of
reporter genes preceded by lexA operators. Similar activity is not observed in the
appended domains of glutaminyl-, isoleucyl-, and methionyl-tRNA synthetase of S.
cerevisiae. Transcriptional activation activity is also not detected in the appended
domains of valyl-tRNA synthetase of the yeasts Candida albicans and
Schizosaccharomyces pombe despite high sequence similarity to that of S. cerevisiae.
To our knowledge, this appears to be the first example in eukaryotes wherein an
appended domain of a tRNA synthetase contains a transcriptional activation activity.
Further studies are under way to elucidate possible non-canonical functions of
valyl-tRNA synthetase in vivo.

關鍵字(中)

★ tRNA合成酶
★ 轉錄活化

關鍵字(英)

★ transcriptional activation
★ tRNA synthetase

論文目次

目錄
目錄 I
表、圖目錄 IV
縮寫檢索表 V
第一章緒論 1
一、簡介tRNA 合成酶 1
(1)、tRNA 合成酶的生化功能 1
(2)、tRNA 合成酶的分類 1
二、酵母菌中tRNA 合成酶的分佈 2
(1)、蛋白質在哪裡合成？ 2
(2)、要如何由一個基因得到兩個蛋白質產物？ 3
三、簡介Valyl-tRNA synthetase 4
(1)、ValRS 的生化特性 4
(2)、酵母菌中的ValRS 5
四、實驗目的 7
第二章材料與方法 8
一、使用之菌株、載體及培養基 8
二、大腸桿菌之形質轉換 (Transformation) 9
三、酵母菌之形質轉換 (Transformation) 10
四、質體之建構 12
五、互補試驗（Complementation） 13
六、西方墨點法 (Western blotting) 14
七、蛋白質純化 15
八、ValRS 附加區段的細胞核標的訊號測試 18
九、轉錄活化活性測試 19
十、以酵母菌ValRS 附加區段為餌進行two-hybrid 篩選 20
第三章結果 22
一、ValRS 的附加區段不是ValRS 執行細胞功能絕對必須的 22
二、ValRS 附加區段的lysine rich cluster 對酵素in vitro 的胺醯化活性影響不大 23
三、ValRS 附加區段有將蛋白質送入細胞核的特性 24
四、探討附加區段的轉錄活化區 25
五、其他aaRS 的附加區段不具轉錄活化功能 26
六、酵母菌ValRS 附加區段的轉錄活化功能在相近物種之間的ValRS 之附加區段之中是獨特的 28
七、用two-hybrid 的方式篩選會和ValRS 附加區段作用的蛋白質 29
第四章討論 31
參考文獻 35
表 41
圖 43
附錄 59

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謝佳容（2002）酵母菌valyl-tRNA synthetase 附加區段的
生物功能之探討. 中央大學生命科學研究所碩士論文.

指導教授

王健家(Chien-Chia Wang)

審核日期

2005-7-14

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