Chang Chia-Pei, Lin Grace, Chen Shun-Jia, Chiu Wen-Chih, Chen Wen-Heng, Wang Chien-Chia
Department of Life Science, National Central University, Jung-li, 32001 Taiwan.
J Biol Chem. 2008 Nov 7;283(45):30699-706. doi: 10.1074/jbc.M805339200. Epub 2008 Aug 28.
Previous studies showed that valyl-tRNA synthetase of Saccharomyces cerevisiae contains an N-terminal polypeptide extension of 97 residues, which is absent from its bacterial relatives, but is conserved in its mammalian homologues. We showed herein that this appended domain and its human counterpart are both nonspecific tRNA-binding domains (K(d) approximately 0.5 microm). Deletion of the appended domain from the yeast enzyme severely impaired its tRNA binding, aminoacylation, and complementation activities. This N-domain-deleted yeast valyl-tRNA synthetase mutant could be rescued by fusion of the equivalent domain from its human homologue. Moreover, fusion of the N-domain of the yeast enzyme or its human counterpart to Escherichia coli glutaminyl-tRNA synthetase enabled the otherwise "inactive" prokaryotic enzyme to function as a yeast enzyme in vivo. Different from the native yeast enzyme, which showed different affinities toward mixed tRNA populations, the fusion enzyme exhibited similar binding affinities for all yeast tRNAs. These results not only underscore the significance of nonspecific tRNA binding in aminoacylation, but also provide insights into the mechanism of the formation of aminoacyl-tRNAs.
先前的研究表明，酿酒酵母的缬氨酰 - tRNA合成酶含有一个由97个残基组成的N端多肽延伸序列，其细菌同源物中不存在该序列，但在其哺乳动物同源物中保守。我们在此表明，这个附加结构域及其人类对应物都是非特异性tRNA结合结构域（解离常数K(d)约为0.5微摩尔）。从酵母酶中删除附加结构域会严重损害其tRNA结合、氨酰化和互补活性。这种缺失N结构域的酵母缬氨酰 - tRNA合成酶突变体可以通过融合其人类同源物的等效结构域来挽救。此外，将酵母酶或其人类对应物的N结构域与大肠杆菌谷氨酰胺 - tRNA合成酶融合，能使原本“无活性”的原核酶在体内发挥酵母酶的功能。与对混合tRNA群体表现出不同亲和力的天然酵母酶不同，融合酶对所有酵母tRNA表现出相似的结合亲和力。这些结果不仅强调了非特异性tRNA结合在氨酰化中的重要性，还为氨酰 - tRNA形成机制提供了见解。
