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

Nature Structural & Molecular Biology volume 14, pages 272–279 (2007)Cite this article

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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Figure 1: Overall structure of the A. fulgidus SepRS–tRNACys–O-phosphoserine complex.
Figure 2: Recognition mechanism for O-phosphoserine.
Figure 3: Recognition mechanisms for the tRNACys anticodon.
Figure 4: Engineering of SepRS.
Figure 5: Model for the evolution of cysteine coding.

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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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Authors and Affiliations

  1. Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

    Ryuya Fukunaga & Shigeyuki Yokoyama

  2. RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, 230-0045, Yokohama, Japan

    Ryuya Fukunaga & Shigeyuki Yokoyama

Authors
  1. Ryuya Fukunaga
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  2. Shigeyuki Yokoyama
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Contributions

R.F. designed, performed and interpreted experiments and wrote the manuscript. S.Y. interpreted experiments and wrote the manuscript.

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Correspondence to Shigeyuki Yokoyama.

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The authors declare no competing financial interests.

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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