在 Saccharomyces cerevisiae 中， ALA1 可以同時解碼細胞質與粒線體的 alanyl-tRNA synthetase (AlaRS)。此一基因利用最靠近 mRNA 5’端的 AUG (即 AUG1) 轉譯出細胞質異構型，利用 AUG 1 上游的兩個重複密碼 ACG (-25) 與 ACG (-24) 轉譯出粒線體異構型。本論文中，我們選殖了 Candida albicans 的 ALA1 基因 (CaALA1)，並針對這個基因的蛋白質轉譯及細胞內分布做進一步的研究，我們發現 CaALA1 可以同時取代 S. cerevisiae ALA1 基因的細胞質與粒線體功能，然而其基因表達的方式卻非常不同。首先我們發現 CaALA1 只轉錄一條 mRNA ，其 5’端位於 AUG1 上游的 24 個核苷酸處。再者， CaALA1 是利用其 mRNA 上的 AUG1 轉譯出一個同時具有粒線體與細胞質功能的蛋白質，而利用與 AUG1 相距八個密碼的 AUG9 轉譯出一個只具有細胞質功能的蛋白質異構型。進一步將此細胞的細胞質及粒線體分離，結果發現由 AUG1 做出的蛋白質可同時分布於細胞質及粒線體，相對地，由 AUG9 做出的蛋白質則只分布於細胞質，此結果與功能性分析完全一致。點突變的實驗結果顯示 CaALA1 的轉譯可能是透過 Leaky scanning 的方式進行，因此改變 AUG1 的週邊序列不但會影響 AUG1 的轉譯起始效率，也會影響 AUG9 的效率。據我們所知，這是在酵母菌中發現的第一個例子：一個 tRNA synthetase 可以同時跑到兩個不同的胞器中作用。 It was previously shown that the cytoplasmic and mitochondrial activities of alanyl-tRNA synthetase of Saccharomyces cerevisiae are provided by two translational products of ALA1, one initiates at the AUG codon closest to the 5’-end of its mRNA transcripts and the other at upstream in-frame redundant non-AUG codons (i. e., ACG(-25) and ACG(-24)). In this thesis, we report the cloning and characterization of a homologous gene from Candida albicans. Functional assays show that this gene can substitute for both the cytoplasmic and mitochondrial functions of ALA1 in S. cerevisiae; however, several points regarding the pattern of gene expression differ from mechanisms used to regulate expression of ScALA1. First, the results of 5’-RACE showed expression of only a single transcript whose 5’-end mapped to nucleotide position -24 relative to ATG1. However, this single transcript codes for two distinct protein isoforms through alternative initiation from two in-frame AUG triplets 8 codons apart. Complementation assays showed that the AUG1-initiated protein can complement for both cytoplasmic and mitochondrial activities of ScALA1, while the AUG9-initiated protein functions only in the cytoplasm. Fractionation assays also showed that the AUG1-initiated protein form can be partitioned in both compartments; however, the AUG9-initiated protein form was exclusively confined to the cytoplasm. Next, we tested whether the mechanism used by CaALA1 to initiate translation was due to “leaky scanning”. Therefore, a series of point mutations were introduced into the sequence context surrounding the first initiator. Results showed that the efficiencies of translation initiation by the two initiators could be influenced by changing the sequence context of AUG1, consistent with the leaky scanning model of translation initiation. To our knowledge, this appears to be the first example in yeast wherein a naturally occurring form of a tRNA synthetase can play roles in both compartments.
