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.
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Acknowledgements
We thank S. Sekine, T. Ito, T. Yanagisawa, A. Shimada, T. Sengoku, R. Ishii, M. Kawamoto, N. Shimizu, and H. Sakai for their help in data collection at SPring-8 and R. Akasaka for help in the analytical ultracentrifugation analysis. This work was supported by Grants-in-Aid for Scientific Research in Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan and by the RIKEN Structural Genomics/Proteomics Initiative of the National Project on Protein Structural and Functional Analyses, MEXT. R.F. was supported by Research Fellowships from the Japan Society for the Promotion of Science for Young Scientists.
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R.F. designed, performed and interpreted experiments and wrote the manuscript. S.Y. interpreted experiments and wrote the manuscript.
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Supplementary information
Supplementary Fig. 1
Structure of M. jannaschii SepRS. (PDF 530 kb)
Supplementary Fig. 2
Structure of A. fulgidus SepRS. (PDF 609 kb)
Supplementary Fig. 3
tRNA anticodon loop conformations. (PDF 382 kb)
Supplementary Fig. 4
Structure of the suppressor complexes of A. fulgidus SepRS. (PDF 1200 kb)
Supplementary Fig. 5
Engineering of A. fulgidus SepRS. (PDF 41 kb)
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Fukunaga, R., Yokoyama, S. Structural insights into the first step of RNA-dependent cysteine biosynthesis in archaea. Nat Struct Mol Biol 14, 272–279 (2007). https://doi.org/10.1038/nsmb1219
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DOI: https://doi.org/10.1038/nsmb1219
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