Study of yeast mitochondrial glutamyl-tRNA synthetase

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Title:	Study of yeast mitochondrial glutamyl-tRNA synthetase
Authors:	林楨桓;Lin,Chen-huan
Contributors:	生命科學系
Keywords:	tRNA合成酵素;粒線體;酵母菌;附加區段;Appended domain;mitochondrial;tRNA synthetase;yeast
Date:	2012-12-26
Issue Date:	2013-01-29 14:40:20 (UTC+8)
Publisher:	國立中央大學
Abstract:	早期研究指出酵母菌valyl-tRNA synthetase (ValRS) 附加區段 (1-98) 具有非專一性的tRNA 鍵結活性 (Kd ~2 uM)，此活性具有幫助aminoacylation的功能。而ValRS 的N端附加區段 (1-135) 融合到DNA結合蛋白LexA形成一個轉錄活化因子且其轉錄活性來自附加區段內的五個胺基酸 (WYDWW)。使用這五個胺基酸當作基礎，衍生出轉錄活性更強的7個色胺酸 (W7)，它的活性是原本五個胺基酸的3倍。我們希望經由此研究更了解轉錄活化區段的一些生化特性及對轉錄活化機制有新的認識。經過數十年的研究，對於aminoacyl-tRNA synthetases (aaRSs)的結構與功能的理解已有長足進展。相較之下，對於他們的演化路徑仍然相當模糊，尤其是具有雙功能之基因，例如非鑑別的glutamyl-tRNA synthetase (non-discriminating GluRS; ND-GluRS) 它可同時將Glu 接到tRNAglu 或tRNAGln上。藍綠菌的GluRS 就是一個ND-GluRS它可以同時辨認tRNAglu (形成Glu-tRNAglu) 及tRNAgln (形成Glu-tRNAgln)，我們發現這個 ND-GluRS可以取代酵母菌MSE1 基因 (轉譯出粒線體的GluRS ) 的功能，而粒線體 GluRS是一個鑑別的酵素，只能形成Glu-tRNAglu ，在 GluRS分子演化上這是一個有趣的發現。The earlier studies suggested that the Ad of yeast valyl-tRNA synthetase (ValRS) (residues 1~98) possesses non-specific tRNA-binding activity (with a Kd of ~2 μM) that significantly contributes to tRNA binding and aminoacylation activities of the enzyme. The N-terminal domain (residues 1~135) of yeast ValRS can act as an transcription activation domain (AD) when fused to a sequence-specific DNA-binding domain (DBD) LexA, and this transcriptional activity is mainly attributed to a pentapeptide (WYDWW) close to the C-terminus of the N domain. Using this pentapeptide as a reference, a much more active peptide, heptatryptophan (W7), was devised. W7 produced activity ~3-fold higher than that of the native pentapeptide or the N domain. It is our hope that information obtained in this study will advance our understanding of the biochemical properties of an AD in general, and also provide new insights into the mechanisms of transcription activation in particular. After decades of investigation, knowledge of structure and functions of aminoacyl-tRNA synthetases (aaRSs) have progressed by leaps and bounds. However, their evolution paths are still obscure, especially for the dual-functional AARSs such as non-discriminating GluRS (ND-GluRS). It can charge both tRNAGlu and tRNAGln with Glu. Syncchocystis 6803 GluRS (SyGluRS) is an ND-GluRS enzyme that functions on its cognate tRNAGlu (Glu-tRNAGlu) and on noncognate tRNAGln.(Glu-tRNAGln). We find the ND-GluRS that it could rescue the growth defects of the GluRSm knockout strains (CSJ2). The finding might provide insights into the evolution of ND-GluRS and D-GluRS.
Appears in Collections:	[Graduate Institute of Life Science] Electronic Thesis & Dissertation

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