酵母菌的可誘導GlyRS之特性

Characterization of an inducible yeast glycyl-tRNA synthetase gene

吳怡樺(Yi-Hua Wu)

指導教授 ： 王健家 ； 王紹文

國立中央大學/理學院/生命科學系/博士(2015年)

摘要

Aminoacyl-tRNA synthetases(簡稱aaRSs)其催化生成aminoacyl-tRNAs，提供蛋白質生合成的原料。它們是一群不可或缺且古老的酵素家族，存在於自然界所有的生物體內，正因如此，我們便可利用它們做演化研究的依據。之前的研究指出，酵母菌(Saccharomyces cerevisiae)的細胞核內有編碼兩種不同的GlyRS基因，分別為GRS1基因(解碼glycyl-tRNA synthetase1，簡稱GlyRS1)和GRS2基因(解碼glycyl-tRNA synthetase2，簡稱GlyRS2)。其中蛋白質GlyRS1，即具有細胞質和粒線體的功能，且與GlyRS2做蛋白質序列比較發現明顯地多了一段嵌入肽鏈(insertion peptide) ，此外，兩種蛋白質在結構和序列上並無明顯差異；而GRS2基因平常於酵母菌體內不表現，類似「假基因(Pseudogene)」，其看似演化留下的痕跡(在高等真核生物並無此基因，且所有已知酵母菌菌株中，只有S. cerevisiae和V. polyspora具有GRS2基因)，但我們發現若以生物技術所獲得其表現的蛋白質GlyRS2，竟然具有催化活性，因此其所扮演的角色及生理意義，遂成為我們研究的終極目的。首先，我們發現GRS2基因在一些極端條件下(高溫、存在酒精、氧化壓力等)，會被誘導使表現量上升，且由實驗結果可知其蛋白質GlyRS2在高溫條件具有較佳的活性及穩定性；接著，藉由從各種不同面向的生物體內和體外實驗結果證明「嵌入肽鏈」對GlyRS1重要性，並與GlyRS2做比較，再更進一步討論GlyRS1和GlyRS2之間演化上的關係；最後，我們由體外實驗結果發現GlyRS2於酸性和存在酒精條件下，催化活性並無明顯變化，反而GlyRS1催化活性大受影響，此結果間接解釋了GlyRS2真正的生理功能，以及為何它在演化這條漫漫長路上罕見地被保留至今，其可能與釀酒酵母菌S. cerevisiae的發酵作用有關。

關鍵字

glycyl-tRNA synthetase ； GlyRS1 ； GlyRS2 ； pseudogene-like gene ； stress-inducible gene ； gene duplication ； insertion peptide ； fermentation ； whole-genome duplication

並列摘要

Aminoacyl-tRNA synthetases (aaRSs) participate in producing aminoacyl-tRNAs (aa-tRNAs) which are the precursors in protein synthesis. They are a large and old family of housekeeping enzymes that are pivotal in protein translation and other vital cellular processes in the overall organisms. Thus, aaRSs are useful proteins to study evolution. In previous studies, Saccharomyces cerevisiae possesses two distinct nuclear glycyl-tRNA synthetase (GlyRS) genes, GRS1 and GRS2. GRS1 encodes GlyRS1 which contains both cytoplasmic and mitochondrial activities, while GRS2 (which encodes GlyRS2) is dysfunctional and not required for growth. They are much alike, but are distinguished by an insertion peptide of GlyRS1 that is absent from GlyRS2 and other eukaryotic homologues. In the past, GRS2 was considered as a pseudogene-like gene and only exists in S. cerevisiae and V. polyspora. However, we observed that in vitro purified GlyRS2 had the aminoacylation ability. The exciting results made us curious of the biological function of GlyRS2 in yeast and discuss why GlyRS2 has survived during the GlyRSs’ evolution? First, we herein present evidence that the expression of GRS2 was drastically induced in the some stress conditions (ex: heat shock, alcohol and hydrogen peroxide addition). In addition, GlyRS2 was more stable and more active under heat shock conditions than under normal conditions. Second, we proved that the insertion peptide of GlyRS1 facilitates both productive docking and catalysis of cognate tRNAs in vivo and in vitro. Further, we found that GlyRS2 is an active enzyme essentially resembling the insertion peptide-deleted form of GlyRS1. And a phylogenetic analysis showed that GRS1 and GRS2 are paralogues that arose from a gene duplication event relatively recently. Finally, we found that GlyRS2 has more stable aminoacylation activity than GlyRS1 in the acidic and alcohol-added conditions. The results show the possibly biological function of GlyRS2 in yeast and explain why GlyRS2 is kept during the evolution of GlyRS family.

並列關鍵字

glycyl-tRNA synthetase ； GlyRS1 ； GlyRS2 ； pseudogene-like gene ； stress-inducible gene ； gene duplication ； insertion peptide ； fermentation ； whole-genome duplication

參考文獻

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