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.
