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 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.
