探討酵母菌GlyRS基因的表現及功能

Study of the expression and function of yeast GlyRS genes

陳順佳(Shun-Jia Chen)

指導教授 ： 王健家

國立中央大學/理學院/生命科學系/博士(2011年)

摘要

之前的研究發現釀酒酵母中ALA1及GRS1基因都是使用non-AUG起始密碼轉譯出各自的粒線體型蛋白質。我們實驗室研究發現non-AUG轉譯起始效率大約是AUG的三分之一或更少，而且周邊序列對non-AUG的轉譯效率影響也較對AUG大，除此之外，也發現最好的周邊序列是AAA (-3到-1核甘酸)。重複的non-AUG起始密碼(如ACGACG)通常能增加轉譯的效率；但某些重複的non-AUG起始密碼(如GUGGUG)會使周邊序列變差(尤其是-3核甘酸)，則其轉譯效率反而變差。另一個重大發現是：絕大部分酵母菌都只含有一個glycyl-tRNA synthetase (GlyRS)基因(稱做GRS1)，但是S. cerevisiae及V. polyspora卻有兩個相異的GlyRS基因(稱做GRS1及GRS2)。研究結果顯示：所有的酵母菌GRS1基因都是雙功能的，能同時做出細胞質及粒線體的GlyRS異構型，但是GRS2基因卻是非必需的。然而最令人驚奇的是：GRS2基因雖然在正常培養條件下不表現，它卻可以被一些逆境條件誘導表現，例如鹼性培養基(pH 8.0)或高溫生長條件(37°C)，且純化出來的GlyRS2蛋白質具有相當程度的胺醯化活性。在正常(30°C)及高溫條件下(37°C) GlyRS1與GlyRS2的穩定度都相當高，且在特定高溫條件下GRS2可以互補GRS1的剔除株，維持其正常生長。也許被誘導出來的GlyRS2在某些條件下可以取代GlyRS1的功能，另一種可能性則是GlyRS2參與其它代謝機制。

關鍵字

none

並列摘要

Previous studies showed that ALA1 and GRS1 of Saccharomyces cerevisiae can initiate translation of their respective mitochondrial forms from a non-AUG codon. Our results showed that the translation efficiency of non-AUG initiation is about 30% (or less) relative to that of AUG initiation. In addition, it appeared that a non-AUG initiator codon is much more sensitive to its sequence context than is an AUG initiator codon, and AAA (the nucleotides at position -3 to -1 relative to the initiator) is the most favorable sequence context. Moreover, redundancy of non-AUG initiators, for instance ACGACG, significantly increased the translational efficiency. However, some redundant non-AUG initiators such as UUGUUG that have a poor sequence context (especially at position -3 relative to the second UUG codon), reduced the efficiency of translation. Another interesting discovery reported here was that the majority of yeast species possess a single glycyl-tRNA synthetase (GlyRS) gene (named GRS1). In contrast, S. cerevisiae and Vanderwaltozyma polyspora possessed two GlyRS genes (named GRS1 and GRS2). In all cases, GRS1 was dual-functional, because it encodes both cytoplasmic and mitochondrial forms of GlyRS. In contrast, GRS2 was pseudogene-like and dispensable for growth. Surprisingly, while GRS2 was silent under normal growth conditions (30°C), its expression was induced by certain stresses such as high temperature (37°C) and high external pH (pH 8.0). In addition, purified recombinant GlyRS2 retained a substantial level of aminoacylation activity. Both GlyRS1 and GlyRS2 were appreciably stable in vivo. When overexpressed, the GRS2 gene could rescue the growth defect of a GRS1 knockout strain. Altogether, these data suggest that GRS2 may function to substitute for GRS1 under certain circumstances. Alternatively, it may be involved in other as-yet-unidentified metabolic pathways.

並列關鍵字

aminoacyl-tRNA synthetase ； inducible gene ； sequence context ； aminoacylation

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