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Two enzymes bound to one transfer RNA assume alternative conformations for consecutive reactions

Nature volume 467, pages 612–616 (2010)Cite this article

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Abstract

In most bacteria and all archaea, glutamyl-tRNA synthetase (GluRS) glutamylates both tRNAGlu and tRNAGln, and then Glu-tRNAGln is selectively converted to Gln-tRNAGln by a tRNA-dependent amidotransferase1,2. The mechanisms by which the two enzymes recognize their substrate tRNA(s), and how they cooperate with each other in Gln-tRNAGln synthesis, remain to be determined. Here we report the formation of the ‘glutamine transamidosome’ from the bacterium Thermotoga maritima, consisting of tRNAGln, GluRS and the heterotrimeric amidotransferase GatCAB, and its crystal structure at 3.35 Å resolution. The anticodon-binding body of GluRS recognizes the common features of tRNAGln and tRNAGlu, whereas the tail body of GatCAB recognizes the outer corner of the L-shaped tRNAGln in a tRNAGln-specific manner. GluRS is in the productive form, as its catalytic body binds to the amino-acid-acceptor arm of tRNAGln. In contrast, GatCAB is in the non-productive form: the catalytic body of GatCAB contacts that of GluRS and is located near the acceptor stem of tRNAGln, in an appropriate site to wait for the completion of Glu-tRNAGln formation by GluRS. We identified the hinges between the catalytic and anticodon-binding bodies of GluRS and between the catalytic and tail bodies of GatCAB, which allow both GluRS and GatCAB to adopt the productive and non-productive forms. The catalytic bodies of the two enzymes compete for the acceptor arm of tRNAGln and therefore cannot assume their productive forms simultaneously. The transition from the present glutamylation state, with the productive GluRS and the non-productive GatCAB, to the putative amidation state, with the non-productive GluRS and the productive GatCAB, requires an intermediate state with the two enzymes in their non-productive forms, for steric reasons. The proposed mechanism explains how the transamidosome efficiently performs the two consecutive steps of Gln-tRNAGln formation, with a low risk of releasing the unstable intermediate Glu-tRNAGln.

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Figure 1: Transamidosome formation for Gln-tRNA Gln synthesis.
Figure 2: tRNA Gln outer-corner recognition by the tail body of GatCAB.
Figure 3: Model for the pathway of Gln-tRNA Gln formation.

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Protein Data Bank

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The atomic coordinates and structure factors are deposited in Protein Data Bank under accession numbers 3AKZ and 3AL0.

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Acknowledgements

We thank S. Sekine for discussions and help with data collection, S. Goto-Ito for reading the manuscript, and S. Takahashi, N. Kiyasu and R. Matsunaga for help with sample preparation. We are grateful to the staff of the beam line BL41XU in the SPring-8 and the beam line AR-NE3A in the Photon Factory for their support during data collection. This work was supported by the Targeted Proteins Research Program from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT), a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and MEXT, and the Global COE Program (Integrative Life Science Based on the Study of Biosignaling Mechanisms) from MEXT.

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Authors and Affiliations

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

    Takuhiro Ito & Shigeyuki Yokoyama

  2. Laboratory of Structural Biology, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan ,

    Takuhiro Ito & Shigeyuki Yokoyama

  3. RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama City, Kanagawa 230-0045, Japan ,

    Takuhiro Ito & Shigeyuki Yokoyama

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  1. Takuhiro Ito
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  2. Shigeyuki Yokoyama
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Contributions

T.I. and S.Y. designed the study, T.I. performed the research, and T.I. and S.Y. wrote the paper.

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Correspondence to Shigeyuki Yokoyama.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Discussion, Supplementary Figures 1-9 with legends, Supplementary Table 1, legend for Supplementary Movie 1 and an additional reference. (PDF 3620 kb)

Supplementary Movie 1

The movie shows the transition of the glutamine transamidsome from the glutamylation state to the amidation state – see Supplementary Movie file for full legend. (MOV 9933 kb)

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Ito, T., Yokoyama, S. Two enzymes bound to one transfer RNA assume alternative conformations for consecutive reactions. Nature 467, 612–616 (2010). https://doi.org/10.1038/nature09411

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