Fukunaga Ryuya, Yokoyama Shigeyuki
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan.
Nat Struct Mol Biol. 2007 Apr;14(4):272-9. doi: 10.1038/nsmb1219. Epub 2007 Mar 11.
Cysteine is ligated to tRNA(Cys) by cysteinyl-tRNA synthetase in most organisms. However, in methanogenic archaea lacking cysteinyl-tRNA synthetase, O-phosphoserine is ligated to tRNA(Cys) by O-phosphoseryl-tRNA synthetase (SepRS), and the phosphoseryl-tRNA(Cys) is converted to cysteinyl-tRNA(Cys). In this study, we determined the crystal structure of the SepRS tetramer in complex with tRNA(Cys) and O-phosphoserine at 2.6-A resolution. The catalytic domain of SepRS recognizes the negatively charged side chain of O-phosphoserine at a noncanonical site, using the dipole moment of a conserved alpha-helix. The unique C-terminal domain specifically recognizes the anticodon GCA of tRNA(Cys). On the basis of the structure, we engineered SepRS to recognize tRNA(Cys) mutants with the anticodons UCA and CUA and clarified the anticodon recognition mechanism by crystallography. The mutant SepRS-tRNA pairs may be useful for translational incorporation of O-phosphoserine into proteins in response to the stop codons UGA and UAG.
在大多数生物体中，半胱氨酸通过半胱氨酰 - tRNA合成酶与tRNA（Cys）连接。然而，在缺乏半胱氨酰 - tRNA合成酶的产甲烷古菌中，O - 磷酸丝氨酸通过O - 磷酸丝氨酰 - tRNA合成酶（SepRS）与tRNA（Cys）连接，并且磷酸丝氨酰 - tRNA（Cys）被转化为半胱氨酰 - tRNA（Cys）。在本研究中，我们以2.6埃的分辨率确定了与tRNA（Cys）和O - 磷酸丝氨酸形成复合物的SepRS四聚体的晶体结构。SepRS的催化结构域利用保守α - 螺旋的偶极矩在一个非典型位点识别O - 磷酸丝氨酸带负电荷的侧链。独特的C末端结构域特异性识别tRNA（Cys）的反密码子GCA。基于该结构，我们对SepRS进行工程改造，使其识别具有反密码子UCA和CUA的tRNA（Cys）突变体，并通过晶体学阐明了反密码子识别机制。突变的SepRS - tRNA对可能有助于将O - 磷酸丝氨酸响应终止密码子UGA和UAG翻译掺入蛋白质中。
