古菌中丝氨酰-tRNA合成酶的序列差异
Sequence divergence of seryl-tRNA synthetases in archaea.
作者信息
Kim H S, Vothknecht U C, Hedderich R, Celic I, Söll D
机构信息
Department of Molecular Biophysics and Biochemistry, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8114, USA.
出版信息
J Bacteriol. 1998 Dec;180(24):6446-9. doi: 10.1128/JB.180.24.6446-6449.1998.
The genomic sequences of Methanococcus jannaschii and Methanobacterium thermoautotrophicum contain a structurally uncommon seryl-tRNA synthetase (SerRS) sequence and lack an open reading frame (ORF) for the canonical cysteinyl-tRNA synthetase (CysRS). Therefore, it is not clear if Cys-tRNACys is formed by direct aminoacylation or by a transformation of serine misacylated to tRNACys. To address this question, we prepared SerRS from two methanogenic archaea and measured the enzymatic properties of these proteins. SerRS was purified from M. thermoautotrophicum; its N-terminal peptide sequence matched the sequence deduced from the relevant ORF in the genomic data of M. thermoautotrophicum and M. jannaschii. In addition, SerRS was expressed from a cloned Methanococcus maripaludis serS gene. The two enzymes charged serine to their homologous tRNAs and also accepted Escherichia coli tRNA as substrate for aminoacylation. Gel shift experiments showed that M. thermoautotrophicum SerRS did not mischarge tRNACys with serine. This indicates that Cys-tRNACys is formed by direct acylation in these organisms.
詹氏甲烷球菌和嗜热自养甲烷杆菌的基因组序列含有结构上不常见的丝氨酰 - tRNA合成酶（SerRS）序列，并且缺乏典型的半胱氨酰 - tRNA合成酶（CysRS）的开放阅读框（ORF）。因此，尚不清楚Cys - tRNACys是通过直接氨基酰化形成，还是由错误酰化到tRNACys上的丝氨酸转化形成。为了解决这个问题，我们从两种产甲烷古菌中制备了SerRS，并测定了这些蛋白质的酶学性质。SerRS是从嗜热自养甲烷杆菌中纯化得到的；其N端肽序列与嗜热自养甲烷杆菌和詹氏甲烷球菌基因组数据中相关ORF推导的序列匹配。此外，SerRS是从克隆的沼泽甲烷球菌serS基因表达而来。这两种酶将丝氨酸加载到它们的同源tRNA上，并且也接受大肠杆菌tRNA作为氨基酰化的底物。凝胶迁移实验表明，嗜热自养甲烷杆菌SerRS不会使tRNACys错误地加载丝氨酸。这表明在这些生物体中，Cys - tRNACys是通过直接酰化形成的。
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