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    請使用永久網址來引用或連結此文件: http://ir.lib.ncu.edu.tw/handle/987654321/6293


    題名: 酵母菌轉譯起始機制的研究;Elucidating a novel translation initiation mechanism for a eukaryotic gene
    作者: 唐蕙苓;Huei-Lin Tang
    貢獻者: 生命科學研究所
    關鍵詞: non-AUG 轉譯起始密碼;alternative transcription and translation;leaky scanning;non-AUG initiator
    日期: 2002-06-03
    上傳時間: 2009-09-22 10:17:05 (UTC+8)
    出版者: 國立中央大學圖書館
    摘要: 中文摘要 目前已知在原核及高等真核細胞中,少數的基因可以使用 non-ATG作為轉譯的起始密碼,但在低等真核細胞如酵母菌Saccharomyces cerevisiae卻從未發現過。我們的實驗結果顯示酵母菌只有一個alanyl-tRNA synthetase (AlaRS)的基因 (ALA1),這個基因可以同時轉譯出細胞質及粒腺體所需的AlaRS。然而在這個基因的5’端卻只有一個ATG起始密碼(即ATG1),藉由5’ RACE的方法,我們發現ALA1可以轉錄出三條長短不同的訊息RNA,其5’端分別座落在-143,-105和-54核苷酸上。點突變的結果顯示,ALA1以ATG1 為起始密碼轉譯出細胞質的AlaRS,卻以上游的二個non-AUG (即ACG-25和ACG-24)為起始密碼轉譯出較大的粒腺體AlaRS。弁鄐W這兩個ACG都可以用來作為轉譯起始密碼,但是以ACG-25為較主要的起始密碼。用西方轉漬法來測定其蛋白質的表現,也得到了一致性的結果。而這樣的結果也首次證明了在正常生理條件下酵母菌可以使用non-AUG作為轉譯起始密碼,且這項發現不但銜接了由原核生物到高等真核生物在non-AUG使用上的斷層,也讓我們對酵母菌基因表現的調控有了更深一層的認 Abstract Initiation of protein translation at non-ATG codons has been shown to occur naturally, although rarely in prokaryotes and high eukaryotes, and never in yeast. In this thesis, we provide strong evidence that a non-ATG codon is used as the alternative translational start site of the yeast gene ALA1, which is the only gene in Saccharomyces cerevisiae coding for alanyl-tRNA synthetase. An in vivo functional assay shows that ALA1 is a bifunctional gene that provides both the cytoplasmic and mitochondrial functions. However, unlike most other bifunnctional genes, which contain two alternative in-frame ATG initiators, there is only one ATG codon, designated ATG1, close to the 5’-end of the ALA1open reading frame. Transcriptional mapping reveals the existence of three overlapping transcripts for ALA1, with 5’ ends at positions –143, -105, -54, respectively, relative to the “A” nucleotide of ATG. Site-specific mutagenesis shows that the cytoplasmic and mitochondrial functions of ALA1 are provided by two distinct protein products: a cytoplasmic form initiated at ATG1 and a longer mitochondrial form initiated at two in-frame non-ATG codons, ACG-25 and ACG-24. These two ACG codons function redundantly in initiation of translation. Either one can be functional in the absence of the other; however, the first ACG codon appears to play a predominant role in protein synthesis. Western blot analysis further confirms the initiator function of these two codons. This appears to be the first example in which a non-ATG codon is used physiologically as a transitional initiator in yeast.
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