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Structural basis for translational fidelity ensured by transfer RNA lysidine synthetase

Nature volume 461, pages 1144–1148 (2009)Cite this article

Abstract

Maturation of precursor transfer RNA (pre-tRNA) includes excision of the 5′ leader and 3′ trailer sequences, removal of introns and addition of the CCA terminus1,2,3. Nucleotide modifications are incorporated at different stages of tRNA processing, after the RNA molecule adopts the proper conformation. In bacteria, tRNAIle2 lysidine synthetase (TilS) modifies cytidine into lysidine (L; 2-lysyl-cytidine) at the first anticodon of tRNAIle2 (refs 4–9). This modification switches tRNAIle2 from a methionine-specific to an isoleucine-specific tRNA9. However, the aminoacylation of tRNAIle2 by methionyl-tRNA synthetase (MetRS), before the modification by TilS, might lead to the misincorporation of methionine in response to isoleucine codons. The mechanism used by bacteria to avoid this pitfall is unknown. Here we show that the TilS enzyme specifically recognizes and modifies tRNAIle2 in its precursor form, thereby avoiding translation errors. We identified the lysidine modification in pre-tRNAIle2 isolated from RNase-E-deficient Escherichia coli and did not detect mature tRNAIle2 lacking this modification. Our kinetic analyses revealed that TilS can modify both types of RNA molecule with comparable efficiencies. X-ray crystallography and mutational analyses revealed that TilS specifically recognizes the entire L-shape structure in pre-tRNAIle2 through extensive interactions coupled with sequential domain movements. Our results demonstrate how TilS prevents the recognition of tRNAIle2 by MetRS and achieves high specificity for its substrate. These two key points form the basis for maintaining the fidelity of isoleucine codon translation in bacteria. Our findings also provide a rationale for the necessity of incorporating specific modifications at the precursor level during tRNA biogenesis.

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Figure 1: Mass spectrometric analysis of pre- and mature tRNAIle2s purified from E. coli.
Figure 2: tRNA recognition by Gk TilS.
Figure 3: Sequential tRNA recognition mechanism.

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Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession numbers 3A2K (GkTilS–tRNA complex) and 3HJ7 (ASB domain).

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Acknowledgements

We thank the beamline staff at BL41XU of SPring-8 (Harima, Japan) and NW12 of PF-AR (Tsukuba, Japan) for help during data collection. We thank H. Aiba for providing the temperature-sensitive E. coli strain of RNase E, A. Soma and Y. Sekine for discussions, and T. Suzuki and Y. Sakaguchi for support with mass spectrometry. This work was supported by a SORST program grant from Japan Science and Technology to O.N., by a grant for the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to O.N. and T.S., by grants from MEXT to R.I. and O.N., and by Mitsubishi Foundation and Kurata Memorial Hitachi Science and Technology Foundation grants to O.N. L.B. and K.N. were supported by a postdoctoral fellowship for foreign researchers and a young scientist fellowship, respectively, from the Japan Society for the Promotion of Science.

Author Contributions K.N. performed purification, crystallization and structure determination of the complex, and biochemical analyses. L.B. performed purification, crystallization and structure determination of the ASB domain, and biochemical analyses. S.K. purified the native tRNAs and performed the mass spectrometry analysis under the supervision of T.S. R.I. performed the molecular dynamics analysis and assisted with the structural determination. O.N. assisted with the structure determination. All authors discussed the results and commented on the manuscript. O.N. supervised all the work.

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Author notes

  1. Kotaro Nakanishi

    Present address: Present address: Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.,

Authors and Affiliations

  1. Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Kanagawa 225-8501, Japan,

    Kotaro Nakanishi & Osamu Nureki

  2. Department of Basic Medical Sciences, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan,

    Luc Bonnefond, Ryuichiro Ishitani & Osamu Nureki

  3. Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan,

    Satoshi Kimura & Tsutomu Suzuki

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Correspondence to Osamu Nureki.

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Nakanishi, K., Bonnefond, L., Kimura, S. et al. Structural basis for translational fidelity ensured by transfer RNA lysidine synthetase. Nature 461, 1144–1148 (2009). https://doi.org/10.1038/nature08474

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