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姓名 葉蟬嫻(Chan-Hsien Yeh)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 探討酵母菌ALA1基因的non-AUG轉譯機制
(Mechamism of non-AUG initiation in yeast ALA1)
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摘要(中) ALA1是酵母菌唯一的alanyl-tRNA synthetase (AlaRS) 基因,其5’端只有一個ATG起始密碼 (即ATG1),卻可以轉譯出兩種型的AlaRS,分別作用在細胞質及粒腺體內。ALA1利用ATG1轉譯出細胞質的AlaRS,利用ATG1上游的二個重複non-ATG (即ACG-25和ACG-24) 作出序列較長的粒腺體AlaRS。本實驗著重在進一步研究酵母菌的轉譯機制,已知ALA1可以轉錄出三條長短不同的messenger RNA (簡稱mRNA),其5’端分別座落在核苷酸-143,-105和-54上;利用功能性互補試驗 (complementaion) 及西方點漬法 (western blotting),我發現ALA1會利用選擇性轉錄及轉譯的方式,由長度不同的mRNA做出大小不同的兩種AlaRS,也可用leaky scanning的方式由同一條mRNA合成細胞質及粒腺體AlaRS,一般而言這兩種機制甚少同時出現,因此ALA1是相當獨特的例子。除此之外,我也證實non-ATG下游的二級結構,對於non-ATG轉譯機制並非絕對需要,當此結構被破壞時,non-ATG依舊可以有效的產生粒腺體AlaRS,維持酵母菌的生
摘要(英) It was recently shown that ALA1, the only gene in Saccharomyces cerevisiae coding for alanyl-tRNA synthetase (AlaRS), encodes both cytoplasmic and mitochondrial forms. The former is translationally initiated at the ATG codon (designated ATG1) at the 5’-end of its open reading frame, while the latter is initiated from upstream in-frame redundant non-ATG codons (i.e., ACG-25 and ACG-24). In this thesis, I investigated the translational mechanism of ALA1 by which long and short protein isoforms were produced from the single gene. Like many known non-ATG initiators, a secondary structure is identified downstream of ACG-25. However, mutations that destroy the secondary structure do not impair its initiating activity. Functional tests, in combination with Western blot analysis, suggest that the isoforms of AlaRS can be translated from long and short transcripts by alternative transcription/translation, or from a single transcript by leaky scanning. To our knowledge, this appears to be a novel case where both leaky scanning and alternative transcription/translation are involved in the production of protein isoforms.
關鍵字(中) ★ 酵母菌
★ 轉譯機制
關鍵字(英) ★ ALA1
★ AlaRS
★ non-AUG translation
論文目次 目錄……………………………………………………………..….....I
縮寫檢索表…………………………………………………….…....Ⅲ
中文摘要……………………………………………………….…......1
英文摘要……………………………………………………………...2
第一章 緒論………………………………………………………...3
第二章 材料與方法
l 使用之菌株、載體....………………………………………..…..8
l 建構質體……………………………………………………..…..8
l 用PCR方式破壞pseudoknot 二級結構……………..……….9
l 功能性互補試驗 (Complementation ) ―
細胞質AlaRS的功能測試……………………………..……..10
l 功能性互補試驗 ― 粒腺體AlaRS的功能測試………….…12
l 西方點漬法 (Western Blotting)
A. 蛋白質製備 (Protein Preparation) …………….…..….....13
B. 西方點漬法……………………………………………..….15
l 5’ RACE (Rapid Amplification of cDNA Ends) ……….….…17
第三章 結果
I. ALA1基因可以選擇性轉錄及轉譯方式來產生
粒腺體及細胞質兩種型的AlaRS…………….……..……...20
II. ALA1基因可以leaky scanning的方式由同一條
mRNA同時轉譯粒腺體及細胞質AlaRS…………….......21
III. 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……………............22
Ⅳ. 以西方點漬法證實non-AUG可以做為轉譯起始點.…....23
第四章 討論………………………………………………………26
第五章 參考文獻…………………………………………………29
圖表…………………………………………………………………35
附錄一………………………………………………………………41
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指導教授 王健家(Chien-Chia Wang) 審核日期 2003-7-21
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