Structural insights into the first step of RNA-dependent cysteine biosynthesis in archaea

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Abstract
Cysteine is ligated to tRNACys by cysteinyl-tRNA synthetase in most organisms. However, in methanogenic archaea lacking cysteinyl-tRNA synthetase, O-phosphoserine is ligated to tRNACys by O-phosphoseryl–tRNA synthetase (SepRS), and the phosphoseryl-tRNACys is converted to cysteinyl-tRNACys. In this study, we determined the crystal structure of the SepRS tetramer in complex with tRNACys and O-phosphoserine at 2.6-Å 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 α-helix. The unique C-terminal domain specifically recognizes the anticodon GCA of tRNACys. On the basis of the structure, we engineered SepRS to recognize tRNACys 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.