酵母菌ALA1基因的表現調控機制

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姓名

陳念谷(Nian-Ku Chen) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

酵母菌ALA1基因的表現調控機制
(The expression and regulation of the yeast gene,ALA1.)

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★ 探討酵母菌Valyl-tRNA synthetase的生化活性	★ 酵母菌轉譯起始機制的研究

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摘要(中)

中文摘要
截至目前為止，GRS1是酵母菌 (Saccharomyces cerevisiae)唯一已證實可以在自然生理條件下利用non-AUG作為轉譯起始點的基因。最近我們實驗室發現了第二個例子，ALA1是酵母菌中唯一的alanyl-tRNA synthetase (AlaRS) 基因，可同時轉譯出兩個具有功能的異構性蛋白質:一個轉譯自AUG1，另一個則轉譯自上游相同讀碼框的ACGACG密碼 (即ACG-25ACG-24) ; 來自AUG1的異構性蛋白質作用於細胞質內，來自ACG-25ACG-24的則作用於粒腺體中。雖然這兩個蛋白質具有幾乎相同的胺基酸序列，但因為存在的胞器不同，故在功能上不能互相取代 (唐惠苓論文，2002)。在本篇論文我希望進一步分析其轉錄及轉譯機制。由 5’RACE(rapid amplification of 5’cDNA ends)的結果得知ALA1可同時轉錄出三條長短不同的訊息RNA，其5’端分別座落在AUG1上游的117、105和54核苷酸上；利用西方點漬法 (western blotting)的分析，我證實ALA1的確可以使用AUG1及其上游的兩個重複ACG密碼作為轉譯起始點做出兩個大小不同的蛋白質，AUG-25的轉譯效率約為non-AUG的3倍。當破壞ACG-25下游的二級結構時，其轉譯效率幾乎不受影響，因此推測這個二級結構對於non-AUG轉譯機制可能並非絕對需要。我們也意外發現UUG-16 及 AUA-1可能也可以充當轉譯起始點做出極微量的蛋白質。以上這些結果顯示利用non-AUG為轉譯起始點的機制可能分佈很廣泛。

摘要(英)

Abstract
So far, only the GRS1 gene has been reported to use a naturally occurring non-AUG triplet as the translation initiator in Saccharomyces cerevisiae. Recently, our lab has discovered a second example of non-AUG initiation in the yeast. ALA1 is the only gene in Saccharomyces cerevisiae (alanyl-tRNA synthetase, AlaRS), encodes both cytoplasmic and mitochondrial forms of the enzyme. The former is translationally initiated at the AUG codon (designated as AUG1) close to the 5’-end of its open reading frame, while the latter is initiated from upstream in-frame redundant non-AUG codons (i.e., ACG-25 and ACG-24). In this thesis, I investigated the transcriptional and translational profiles of ALA1, aiming at elucidating the mechanism that is responsible for the bifunctional phenotype of this gene. Transcriptional mapping reveals the existence of three overlapping transcripts for ALA1, with 5’ ends at positions 117, 105, and 54, respectively, relative to the “A” nucleotide of AUG1. Western blot analysis further confirms the initiating activity of the redundant ACG codons. The frequency of initiation at ACG-25/ACG-24 is around 30% relative to that at AUG-25. Results also suggest that certain non-AUG codons in the presequence, such as UUG-16 and AUA-1, are also involved in the synthesis of minor protein isoforms. Unexpectedly, mutations that destroyed the pseudoknot structure, downstream of the redundant ACG codons, did not impair this initiating activity. Taken together, our results suggest that non-AUG initiation is a mechanism more widespread than previously thought.

關鍵字(中)

★ 轉譯
★ 非-AUG
★ 酵母菌
★ ALA1

關鍵字(英)

★ ALA1
★ translation
★ non-AUG
★ Yeast

論文目次

主目錄
中文摘要………………………………………………………..….I
英文摘要…………………………………………………………...II
誌謝...................................................................................................III
主目錄…………………………………………………………….. IV
圖目錄...............................................................................................VI
縮寫檢索表………………………………………………………..VII
第一章緒論………………………………………………….…..1
第二章材料與方法
? 使用之菌株、載體....………………………………….…6
? 建構質體……………………………………………….…6
? 用點突變方式破壞pseudoknot 二級結構……………..7
? 5’ RACE (Rapid Amplification of cDNA Ends)….…....8
? 西方點漬法 (Western Blotting)
A. 蛋白質製備………………………….………….…..10
B. 西方點漬法………………………………………....11
? 半乳糖苷酶定量及酵素活性分析……………………...13
第三章結果
一、ALA1基因轉錄出多條訊息RNA………………............14
二、用西方點漬法證明重複的ACG密碼可以做
為轉譯起點……..............…..............................................15
三、破壞mRNA上的pseudoknot二級結構，對於
粒腺體蛋白質的轉譯並無明顯的影響..............................18
四、ACG起始密碼的點突變不會改變ALA1 mRNA
的表現量…………………..………..………………….….18
五、non-AUG半乳糖苷酶酵素活性分析的結果與
西方點漬法類似……………………………………….….19
第四章討論…………………………………………….…….…20
第五章參考文獻……………………………………………..…38
附錄一………………………………………….……….…….…...44
plasmid list and primer list………………..…..……….….….….45
圖目錄
圖1.aaRS的基本生化功能…………………………….….....23
圖2.選擇性轉錄及轉譯機制……………………………..…..24
圖3.鑑定ALA1 mRNA的5端…………………………..….25
圖4.以西方點墨法觀察由non-AUG轉譯的蛋白質情形….27
圖5.RT-PCR 測定mRNA的表現量及穩定性………….…31
圖6.半乳糖苷酶酵素活性分析定量non-AUG轉譯效率….35
圖7.ALA1基因pseudoknot二級結構的構造及被破壞後
RNA示意圖…...……………...………………………....37

參考文獻

第五章參考文獻
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指導教授

王健家(Chien-Chia Wang)

審核日期

2004-7-17

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