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Snapshots of tRNA sulphuration via an adenylated intermediate

Nature volume 442, pages 419–424 (2006)Cite this article

Abstract

Uridine at the first anticodon position (U34) of glutamate, lysine and glutamine transfer RNAs is universally modified by thiouridylase into 2-thiouridine (s2U34), which is crucial for precise translation by restricting codon–anticodon wobble during protein synthesis on the ribosome. However, it remains unclear how the enzyme incorporates reactive sulphur into the correct position of the uridine base. Here we present the crystal structures of the MnmA thiouridylase–tRNA complex in three discrete forms, which provide snapshots of the sequential chemical reactions during RNA sulphuration. On enzyme activation, an α-helix overhanging the active site is restructured into an idiosyncratic β-hairpin-containing loop, which packs the flipped-out U34 deeply into the catalytic pocket and triggers the activation of the catalytic cysteine residues. The adenylated RNA intermediate is trapped. Thus, the active closed-conformation of the complex ensures accurate sulphur incorporation into the activated uridine carbon by forming a catalytic chamber to prevent solvent from accessing the catalytic site. The structures of the complex with glutamate tRNA further reveal how MnmA specifically recognizes its three different tRNA substrates. These findings provide the structural basis for a general mechanism whereby an enzyme incorporates a reactive atom at a precise position in a biological molecule.

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Figure 1: Structure of the MnmA–tRNA Glu complex.
Figure 2: tRNA recognition.
Figure 3: Sequential snapshots of the sulphuration reaction.
Figure 5: Catalytic mechanisms of MnmA.
Figure 4: In vitro sulphur transfer from IscS to tRNAGlu.

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Acknowledgements

We thank M. Ibba for helpful improvement of the manuscript. We thank M. Kawamoto and N. Shimizu for their help in data collection at SPring-8. This work was supported by a PRESTO program grant from Japan Science and Technology (JST) to O.N., 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., grants from MEXT to O.N., S.F. and T.S., a JSPS Fellowship for Japanese Junior Scientists to Y.I., the Asahi Glass Foundation to S.F., and Sumitomo and Kurata Memorial Hitachi Science and Technology Foundation grants to O.N. Author Contributions T.N. carried out structural determination and mutant preparation, and wrote the paper with editing from S.F. and O.N. S.F. and O.N. assisted the structural determination. Y.I. carried out the biochemical analyses under supervision of T.S. All authors discussed the results and commented on the manuscript. O.N. supervised all of the work.

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Authors and Affiliations

  1. Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Kanagawa, 226-8501, Yokohama-shi, Japan

    Tomoyuki Numata, Shuya Fukai & Osamu Nureki

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

    Yoshiho Ikeuchi & Tsutomu Suzuki

  3. PRESTO, JST, Honcho, Kawaguchi-shi, Saitama, 332-0012, Japan

    Osamu Nureki

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

    Osamu Nureki

Authors
  1. Tomoyuki Numata
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Correspondence to Osamu Nureki.

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Coordinates and structure factors are deposited in the Protein Data Bank under accession code 2DER, 2DET and 2DEU for the initial tRNA-binding form, the prereaction form, and the adenylated intermediate form, respectively. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1–6 (PDF 60690 kb)

Supplementary Table 1

This table shows the data collection and refinement statistics. (PDF 86 kb)

Supplementary Notes

This file contains Supplementary Discussions, Supplementary Methods, Supplementary Figure Legends and additional references. (DOC 222 kb)

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Numata, T., Ikeuchi, Y., Fukai, S. et al. Snapshots of tRNA sulphuration via an adenylated intermediate. Nature 442, 419–424 (2006). https://doi.org/10.1038/nature04896

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