| 摘要: | 先前研究指出酵母菌的一些tRNA synthetase有一富含離胺酸的附加區段，但是在相對應的大腸桿菌酵素中則無，推測此附加區段可能和tRNA結合有關。酵母菌的valyl-tRNA synthetase (ValRS)中也含有N端的附加區段。我們發現這個附加區段具有非專一性的tRNA鍵結能力，刪除此附加區段會影響酵素的tRNA鍵結能力、胺醯化活性、及互補能力，由此可知附加區段對酵素的重要性。如文獻所知，酵母菌使用non-AUG作為轉譯起始點的例子仍不多，我們發現除了AAG及AGG外，其他和AUG只差一個核苷酸的密碼皆可能作為轉譯起始密碼，除此之外我們發現不同的non-AUG轉譯起始密碼可能偏好不同的周圍序列。酵母菌的ALA1基因使用AUG和non-AUG作為轉譯起始密碼同時做出細胞質及粒線體的alanyl-tRNA synthetase (AlaRS)異構酵素，我們想知道其他酵母菌是否也利用類似機制做出AlaRS。結果發現除了Vanderwaltozyma polyspora以外其他酵母菌也都是利用一個基因同時做出細胞質及粒線體的AlaRS異構型，然而在V. polyspora的細胞核中則有兩個不同的AlaRS基因，一個做細胞質酵素，另一個則做粒線體酵素。演化分析發現所有酵母菌的AlaRS都源自於粒線體，因此推測V. polyspora的AlaRS基因可能經歷至少兩次的基因重組，首先源自粒線體的基因獲得細胞質功能成為雙重功能的基因，而其同源基因（源自古生菌）隨即消失，接著雙重功能的基因又經歷全基因體的複製，產生兩個雙功能的基因，最後其中一個保留細胞質功能，另一個則保留粒線體功能。 Previous studies show that many yeast aminoacyl-tRNA synthetases (aaRSs) contain a lysine-rich polypeptide extension or appended domain, which is absent from their bacterial relatives. Yeast valyl-tRNA synthetase (ValRS) possesses an N-terminal appended domain. This domain acts as an auxiliary tRNA-binding domain. Deletion of this domain from ValRS impaired its tRNA-binding, aminoacylation, and complementation activities. This finding underscores the necessity of an appended domain for functioning of a yeast aaRS. So far, only a few examples of non-AUG initiation have been identified in yeast. Our results show that, except for AAG and AGG, all triplets that differ from AUG by a single nucleotide were able to serve as an initiator in yeast. Moreover, each non-AUG initiator codon appeared to have its own favorite sequence context. In yeast, the ALA1 gene encodes the cytoplasmic and mitochondrial isoforms of AlaRS through alternative initiation from an AUG and an upstream non-AUG codon. Interestingly, except for Vanderwaltozyma polyspora, all yeast species studied contained a single dual-functional AlaRS gene. In contrast, V. polyspora contained two distinct nuclear AlaRS genes, one specifying the cytoplasmic form and the other its mitochondrial counterpart. A phylogenetic analysis revealed that all yeast AlaRS genes, including those in V. polyspora, are of mitochondrial origin. These and other results imply that the AlaRS genes in V. polyspora underwent a major genetic recombination at least twice. First, the mitochondrion-type gene gained cytoplasmic function and became a dual-functional gene, while its orthologue was lost. Later, the dual-functional gene of mitochondrial origin was duplicated into two copies during the whole-genome duplication event, each retaining a single function (cytoplasmic or mitochondrial). |
