Structural insights into the first step of RNA-dependent cysteine biosynthesis in archaea
Structural insights into the first step of RNA-dependent cysteine biosynthesis in archaea
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DOI：
10.1038/nsmb1219
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发表时间：
2007-04-01
影响因子：
16.8
通讯作者：
Yokoyama, Shigeyuki
中科院分区：
文献类型：
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作者：
Fukunaga, Ryuya;Yokoyama, Shigeyuki
在大多数生物中，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-磷酸丝氨酸-tRNA合成酶（SepRS）与tRNA（Cys）连接，并且磷酸丝氨酸-tRNA（Cys）转化为半胱氨酰-tRNA（Cys）。在这项研究中，我们确定了SepRS四聚体与tRNA（Cys）和O-磷酸丝氨酸复合物的晶体结构，分辨率为2.6埃。SepRS的催化结构域利用保守的α-螺旋的偶极矩在非典型位点识别O-磷酸丝氨酸的带负电荷的侧链。独特的C-末端结构域特异性识别tRNA的反密码子GCA（Cys）。在此基础上，我们设计了SepRS，使其能够识别含有反密码子UCA和CUA的tRNA（Cys）突变体，并通过晶体学方法阐明了其反密码子识别机制。突变的SepRS-tRNA对可用于响应于终止密码子UGA和UAG将O-磷酸丝氨酸翻译掺入蛋白质中。
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-angstrom 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.