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A paralog of lysyl-tRNA synthetase aminoacylates a conserved lysine residue in translation elongation factor P

Nature Structural & Molecular Biology volume 17, pages 1136–1143 (2010)Cite this article

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Abstract

Aminoacyl-tRNA synthetase (aaRS) paralogs with unknown functions exist in various species. We now report novel 'protein lysylation' by an Escherichia coli lysyl-tRNA synthetase paralog, GenX/PoxA/YjeA. X-ray crystallographic analysis shows that the structure of the GenX protein resembles that of a class II aaRS. Further in vitro studies reveal that it specifically aminoacylates EF-P with lysine. The shape of the protein substrate mimics that of the L-shaped tRNA, and its lysylation site corresponds to the tRNA 3′ end. Thus, we show how the aaRS architecture can be adapted to achieve aminoacylation of a specific protein. Moreover, in vivo analyses reveal that the translation elongation factor P (EF-P) lysylation by GenX is enhanced by YjeK (lysine 2,3-aminomutase paralog), which is encoded next to the EF-P gene, and might convert α-lysyl–EF-P to β-lysyl–EF-P. In vivo analyses indicate that the EF-P modification by GenX and YjeK is essential for cell survival.

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Figure 1: GenX, a LysRS-like protein, binds a lysyl–AMP analog.
Figure 2: Structure of GenX.
Figure 3: Structure of the GenX•EF-P complex.
Figure 4: GenX post-translationally lysylates EF-P.
Figure 5: MS analyses of the modified EF-P.
Figure 6: YjeK promotes the lysyl modification of EF-P in vivo.
Figure 7: Post-translational modifications of eIF5A and EF-P.

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Acknowledgements

We would like to thank the staff of beamline BL41XU at SPring-8 as well as the staff of the BL-5A and AR-NW12 beamlines at the Photon Factory, S. Sekine, T. Ito, R. Fukunaga and T. Sengoku for assisting with the data collection and the structure determination as well as for discussions, T. Kobayashi (RIKEN) for helpful discussions, M. Asanuma, N. Takeuchi, M. Yamaguchi-Hirafuji, A. Urushibata, R. Akasaka, T. Terada, M. Shirouzu and H. Hirota for MS, K. Takada (RIKEN) and C. Naoe (RIKEN) for the preparation of S30 extracts, ribosomes, tRNAfMet, methionyl-tRNA synthetase and methionyl–tRNAfMet formyltransferase, R. Nakajima (RIKEN), M. Aoki (RIKEN), J. Adachi (RIKEN), N. Ohsawa (RIKEN), K. Katsura (RIKEN), T. Terada (RIKEN) and M. Shirouzu (RIKEN) for the E. coli cell-free protein synthesis system, A. Arakawa, T. Kasai and T. Kigawa for assisting with the ITC analysis, K. Yutani for the DSC analysis and A. Ishii and T. Nakayama for clerical assistance. We are grateful to the National BioResource Project (National Institute of Genetics, Japan) for providing the strains from the Keio collection. This work was supported in part by Grants-in-Aid for Scientific Research in Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Targeted Proteins Research Program and the RIKEN Structural Genomics/Proteomics Initiative (RSGI) in the National Project on Protein Structural and Functional Analyses, Ministry of Education, Culture, Sports, Science and Technology of Japan.

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  1. Ryohei Ishii

    Present address: Present address: Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, New York, USA.,

  2. Tatsuo Yanagisawa and Tomomi Sumida: These authors contributed equally to this work.

Authors and Affiliations

  1. RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama, Japan

    Tatsuo Yanagisawa, Tomomi Sumida, Ryohei Ishii, Chie Takemoto & Shigeyuki Yokoyama

  2. Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

    Ryohei Ishii & Shigeyuki Yokoyama

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  1. Tatsuo Yanagisawa
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Contributions

T.S. performed the crystallographic experiments and the structural analysis; T.Y. performed the biochemical experiments and the structural analysis; R.I. assisted with the structural analysis; T.S., T.Y., R.I., C.T. and S.Y. interpreted the experiments and wrote the manuscript; T.Y. and S.Y. designed the studies.

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Correspondence to Shigeyuki Yokoyama.

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Yanagisawa, T., Sumida, T., Ishii, R. et al. A paralog of lysyl-tRNA synthetase aminoacylates a conserved lysine residue in translation elongation factor P. Nat Struct Mol Biol 17, 1136–1143 (2010). https://doi.org/10.1038/nsmb.1889

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