探討一個真核tRNA合成?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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题名:	探討一個真核tRNA合成?的附加區段之轉錄活化活性;A transcriptional activation domain in a eukaryotic tRNA synthetase
作者:	林明琁;Grace Lin
贡献者:	生命科學研究所
关键词:	tRNA合成酶;轉錄活化;transcriptional activation;tRNA synthetase
日期:	2005-07-04
上传时间:	2009-09-22 10:19:20 (UTC+8)
出版者:	國立中央大學圖書館
摘要:	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。 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.
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