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姓名	陳順佳(Shun-Jia Chen)  查詢紙本館藏	畢業系所	生命科學系
論文名稱	探討酵母菌中non-AUG起始點的周邊序列對轉譯起始效率的影響 (Efficiency of a non-AUG initiator is drastically affected by its sequence context in yeast)
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摘要(中)	先前的研究指出，酵母菌中GRS1基因進行轉譯時，能夠分別使用UUG以及下游的AUG密碼作為轉譯起始點。而本篇論文主旨在於證明GRS1基因進行轉譯時，核醣體辨認個別的轉譯起始密碼之機制主要是利用leaky scanning的方式。不同於利用AUG作為轉譯起始點，當使用non-AUG密碼作為轉譯起始點時，其轉譯起始效率受到起始點兩側的核苷酸序列影響很大，其中又以相對於轉譯起始點-1、-2和-3位置的核苷酸影響最大。我們研究發現對於轉譯起始效率，這三個特殊位置最好及最差的核苷酸組合序列分別為A/A/X (X 為 A或G) 和C/G/C。當UUG起始點的 -3 ~ -1核苷酸由AAA改變成CGC時，其轉譯起始效率減少32倍之多，且導致粒線體蛋白質功能的喪失。雖然之前文獻指出AUG轉譯起始密碼對其周邊序列並不敏感，但當我們將其周邊序列 -3 ~ -1由AAA突變成CGC時，其轉譯效率也降低了8倍，這結果顯示，AUG的效率其實也會受到周邊序列的影響。由我們研究結果顯示，在酵母菌中轉譯起始點的周邊序列對於轉譯起始效率比之前研究的結果都還要重要許多。
摘要(英)	Previous studies have shown that translation of the yeast GRS1 gene is alternatively initiated from a UUG and a downstream AUG triplet. Evidence presented here shows that recognition of these two initiators is mainly mediated by a mechanism known as leaky scanning. Unlike an AUG initiator, efficiency of the non-AUG initiator is drastically affected by its flanking sequences. In particular, the nucleotides at its relative positions -1, -2, and -3. A/A/X (X represents A or G) and C/G/C appear to be the most and least favorable sequences at these positions, respectively. Mutation of the native context sequences -3 ~ -1 from AAA to CGC reduces the initiating activity of the UUG initiator up to 32-fold and results in loss of mitochondrial activity. While an AUG initiator is in general unresponsive to context changes, an AAA(-3 ~ -1) to CGC mutation still reduces its initiating activity up to 8-fold under similar conditions. These results suggest that sequence context is more important than previously expected for initiation in yeast.
關鍵字(中)	★ 周邊序列 ★ 轉譯起始密碼 ★ 酵母菌	關鍵字(英)	★ sequence context ★ translation initiator ★ yeast
論文目次	目錄i 圖目錄iii 縮寫檢索表iv 第一章 緒論1 1.1 Aminoacyl-tRNA synthetase (aaRS)的簡介1 1.2 真核細胞內的aminoacyl-tRNA synthetase (aaRS)2 1.3 轉譯起始密碼的選擇4 1.4 研究目的7第二章 材料與方法 92.0 使用之菌株、載體及培養基9 2.1 大腸桿菌勝任細胞的製備與轉型作用10 2.2 酵母菌勝任細胞的製備與轉型作用12 2.3 質體之選殖13 2.4 點突變 (Site-directed Mutagenesis)15 2.5 功能性互補試驗 (Complementation ) ―測試細胞質功能16 2.6 功能性互補試驗 (Complementation ) ―測試粒線體功能17 2.7 蛋白質製備 (Protein Preparation)19 2.8 SDS-PAGE之蛋白質分子量分析20 2.9 西方氏點墨法 (Western blotting)21 第三章 實驗結果23 3.1 利用leaky scanning方式製造異構型的GlyRS23 3.2 偵測周邊序列對於non-AUG起始作用的重要性27 3.3 比較周邊序列的個別核苷酸對轉譯效率的影響29 3.4 比較各種周邊序列對於AUG及non-AUG的影響31 第四章 討論35 4.1 探討酵母菌中使用leaky scanning的作用機制35 4.2 周邊序列對於non-AUG轉譯起始機制的重要性36 4.3 比較哺乳類細胞和酵母菌中周邊序列對轉譯起始的影響38 第五章 參考文獻40 圖45 附錄一61 附錄二62
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指導教授	王健家(chien-chia Wang)	審核日期	2007-7-9
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