探討酵母菌Histidyl-tRNA synthetase的特性

Characterization of yeast Histidyl-tRNA synthetase.

鄭鈺儒(Yu-ju Cheng)

指導教授 ： 王健家

國立中央大學/理學院/生命科學系/碩士(2012年)

摘要

在酵母菌中具有兩套aminoacyl-tRNA synthetases (aaRSs)，一套在細胞質作用，另一套在粒線體作用。然而酵母菌中，只有一個histidyl-tRNA synthetase (HisRS) 基因HTS1，利用兩個不同的起始密碼轉譯出兩個酵素異構型。在本論文中我們研究不同種的酵母菌HTS1基因，結果顯示所有酵母菌都是使用相似的轉譯機制，利用一個基因轉譯出在細胞質和粒線體作用的HisRS。在E. coli tRNAHis 上G-1:C73是一個重要的決定基，而在酵母菌細胞質和粒線體tRNAHis相對位置分別是G-1:A73和G-1:C73。實驗結果顯示E. coli HisRS無法取代酵母菌細胞質HisRS的活性，可是如果將E. coli HisRS送入粒線體之後，就可以互補酵母菌 HTS1剔除株粒線體的功能。此外，我們發現酵母菌 S. cerevisiae HisRS的胺基端具有一段附加區段，L. elongisporous HisRS中間則有一段插入區段，這兩個區段主要影響酵素對tRNA的KM，對於酵素的kcat則只有輕微的影響，因此，我們猜測此附加區段及插入區段可能用來增加整個酵素對tRNA的親和力。

關鍵字

酵母菌 ； 蛋白質合成 ； 胺醯化

並列摘要

In yeast, there are two distinct sets of aminoacyl-tRNA synthetases (aaRSs), one localized in the cytoplasm and the other in mitochondria. Paradoxically, there is only one histidyl-tRNA synthetase (HisRS) gene, HTS1, in the yeast nuclear genome. As it turns out, this gene encodes both cytoplasmic and mitochondrial forms of HisRS through alternative initiation of translation from two in-frame initiator codons. We herein report that a dual-functional phenotype was conserved in the HTS1 genes of other yeast species. While E. coli tRNAHis species contained a G-1:C73 base pair as the major determinant, yeast cytosolic and mitochondrial tRNAHis isoacceptors respectively contained G-1:A73 and G-1:C73 at the same position. Even though E. coli HisRS failed to substitute for the cytoplasmic activity of yeast HTS1, the bacterial enzyme, upon being imported into mitochondria, could rescue growth defect of a S. cerevisiae HTS1 knockout strain. In addition, we found that the N-terminal appended domain of S. cerevisiae HisRS and an insertion domain of L. elogisporous HisRS significantly contributed to KM of the enzyme for tRNAHis, but had relatively minor effect on the kcat. Our study suggests that the appended domain or insertion domain of yeast HisRS acts as an auxiliary tRNA-binding domain to improve the overall tRNA-binding affinity of the enzyme.

並列關鍵字

histidyl-tRNA synthetase ； aminoacylation

參考文獻

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