探討酵母菌Glycyl-tRNA synthetase的嵌入區段對其生化活性的影響;The biological function of the insertion domain of yesat glycyl-tRNA synthetase

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Title:	探討酵母菌Glycyl-tRNA synthetase的嵌入區段對其生化活性的影響;The biological function of the insertion domain of yesat glycyl-tRNA synthetase
Authors:	陳婉甄;Wan-Chen Chen
Contributors:	生命科學研究所
Keywords:	glycyl-tRNA synthetase
Date:	2009-01-13
Issue Date:	2009-09-22 10:20:26 (UTC+8)
Publisher:	國立中央大學圖書館
Abstract:	先前的研究指出，酵母菌中有兩個 Glycyl-tRNA synthetase（GlyRS）基因，分別是 GRS1（做出GlyRS1）和 GRS2（做出GlyRS2），其中 GRS2並非酵母菌生長所必需的；而 GRS1 分別使用 UUG 以及下游的 AUG 密碼作為轉譯起始點，轉譯出粒線體和細胞質的 GlyRS 異構型。GlyRS1 和 GlyRS2 的胺基酸序列之間具有 59％相同性，兩者之間最大差異在於 GlyRS1 比 GlyRS2 多了一段嵌入區段，該區段約由 30 個胺基酸所組成，位於活化區附近，該區與接觸acceptor stem 有關，且富含 Lysine，主要序列是 KKKRKKKVK。本篇論文探討此嵌入區段是否會影響 GlyRS1 的生化活性及生物功能。結果發現刪除或突變 GlyRS1 的嵌入區段對酵母菌生長效率影響不大；但是在體外實驗下，發現刪除此嵌入區段會降低 GlyRS1 的生化活性；另外我們也發現若將 GlyRS2 與 Arc1p 形成一融合蛋白，該融合蛋白可提供細胞質所需的胺醯化活性，在體外實驗下發現Arc1p可增加 GlyRS2 的生化活性，其結果與體內實驗相呼應。在體外情況下，我們利用聚丙烯醯胺親和力共電泳來分析 GlyRS 的 tRNA 鍵結之研究目前仍在持續進行中。 revious studies have shown that in the yeast, two genes (GRS1 and GRS2) encode glycyl-tRNA synthetase (GlyRS1 and GlyRS2, respectively). GRS1 encodes both cytoplasmic and mitochondrial functions, whereas GRS2 is dispensable. GlyRS1 and GlyRS2 share 59% sequence identity. The most obvious difference between the two proteins is a 30-amino acid insertion that is present in GlyRS1. This insertion is found within an active site subdomain that is predicted to contact the acceptor stem of the tRNA substrate. The insertion is characterized by the lysine-rich consensus sequence KKKRKKKVK. In the work described here we focused on the function of the insertion domain in yeast, hoping to explore whether the mutation or deletion of this insertion will affect cytoplasmic or mitochondrial activities of GlyRS1. Evidence presented here shows that mutation and deletion of the insertion domain of GlyRS1 have little effect on its ability to restore a yeast GRS1 knockout strain growth in vivo. On the other hand, mutation or deletion of the insertion domain reduces its aminoacylation activity in vitro. Furthermore, when Arc1p is fused to GlyRS2, the resultant fusion can rescue the cytoplasmic activities of defect of a yeast GRS1 knockout strain. And Arc1p can increase aminoacylation activity of GlyRS2 in-cis in vitro. Further studies are underway to analyze the tRNA-binding ability of GlyRS by using Polyacrylamide Affinity Coelectrophoresis.
Appears in Collections:	[Graduate Institute of Life Science] Electronic Thesis & Dissertation

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