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Structural basis for orthogonal tRNA specificities of tyrosyl-tRNA synthetases for genetic code expansion

Nature Structural & Molecular Biology volume 10, pages 425–432 (2003)Cite this article

Abstract

The archaeal/eukaryotic tyrosyl-tRNA synthetase (TyrRS)–tRNATyr pairs do not cross-react with their bacterial counterparts. This 'orthogonal' condition is essential for using the archaeal pair to expand the bacterial genetic code. In this study, the structure of the Methanococcus jannaschii TyrRS–tRNATyr–L-tyrosine complex, solved at a resolution of 1.95 Å, reveals that this archaeal TyrRS strictly recognizes the C1-G72 base pair, whereas the bacterial TyrRS recognizes the G1-C72 in a different manner using different residues. These diverse tRNA recognition modes form the basis for the orthogonality. The common tRNATyr identity determinants (the discriminator, A73 and the anticodon residues) are also recognized in manners different from those of the bacterial TyrRS. Based on this finding, we created a mutant TyrRS that aminoacylates the amber suppressor tRNA with C34 65 times more efficiently than does the wild-type enzyme.

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Figure 1: Comparison of the sequences of tRNATyr species and TyrRSs among the three primary kingdoms of life.
Figure 2: Structure of the M. jannaschii TyrRS–tRNATyr–tyrosine complex.
Figure 3: Comparison of the overall structures of TyrRSs.
Figure 4: Acceptor arm recognition by TyrRSs.
Figure 5: Anticodon recognition by TyrRSs.
Figure 6: Protein residues proximal to the side chain of L-tyrosine.

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Acknowledgements

We acknowledge the contributions of M. Kawamoto and H. Sakai for the synchrotron data collection at BL41XU in SPring-8 (Harima, Japan). We thank D. Kiga for helpful advice on the steady-state kinetics of the aminoacylation. We also thank K. Ohtake and M. Takahashi for help in the construction of the TyrRS expression system and R. Ishii for help in the data collection at BL41XU in SPring-8. This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan (S.Y. and O.N.) and by the National Project on Protein Structural and Functional Analyses of MEXT (S.Y.) and the Human Frontier Science Program (O.N. and S.C.).

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Authors and Affiliations

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

    Takatsugu Kobayashi, Osamu Nureki, Ryuichiro Ishitani, Kensaku Sakamoto & Shigeyuki Yokoyama

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

    Osamu Nureki, Kensaku Sakamoto & Shigeyuki Yokoyama

  3. RIKEN Harima Institute at SPring-8, 1-1-1 Kohto, Mikazuki-cho, Sayo, Hyogo, 679-5148, Japan

    Osamu Nureki & Shigeyuki Yokoyama

  4. PRESTO, Japan Science and Technology Corporation, 1-8-4 Honcho, Kawaguchi, 332-0012, Saitama, Japan

    Osamu Nureki

  5. European Molecular Biology Laboratory, Grenoble Outstation, c/o ILL, 156X, Grenoble, F-38042 Cedex 9, France

    Anna Yaremchuk, Michael Tukalo & Stephen Cusack

  6. Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kiev, 252627 143, Ukraine

    Anna Yaremchuk & Michael Tukalo

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  1. Takatsugu Kobayashi
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Correspondence to Shigeyuki Yokoyama.

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Kobayashi, T., Nureki, O., Ishitani, R. et al. Structural basis for orthogonal tRNA specificities of tyrosyl-tRNA synthetases for genetic code expansion. Nat Struct Mol Biol 10, 425–432 (2003). https://doi.org/10.1038/nsb934

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