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Complete crystallographic analysis of the dynamics of CCA sequence addition

Nature volume 443, pages 956960 (26 October 2006) | Download Citation

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Abstract

CCA-adding polymerase matures the essential 3′-CCA terminus of transfer RNA without any nucleic-acid template. However, it remains unclear how the correct nucleotide triphosphate is selected in each reaction step and how the polymerization is driven by the protein and RNA dynamics. Here we present complete sequential snapshots of six complex structures of CCA-adding enzyme and four distinct RNA substrates with and without CTP (cytosine triphosphate) or ATP (adenosine triphosphate). The CCA-lacking RNA stem extends by one base pair to force the discriminator nucleoside into the active-site pocket, and then tracks back after incorporation of the first cytosine monophosphate (CMP). Accommodation of the second CTP clamps the catalytic cleft, inducing a reorientation of the turn, which flips C74 to allow CMP to be accepted. In contrast, after the second CMP is added, the polymerase and RNA primer are locked in the closed state, which directs the subsequent A addition. Between the CTP- and ATP-binding stages, the side-chain conformation of Arg 224 changes markedly; this is controlled by the global motion of the enzyme and position of the primer terminus, and is likely to achieve the CTP/ATP discrimination, depending on the polymerization stage. Throughout the CCA-adding reaction, the enzyme tail domain firmly anchors the TΨC-loop of the tRNA, which ensures accurate polymerization and termination.

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Acknowledgements

We thank M. Ibba for improvement of the manuscript. We thank the beamline staff at BL41XU of SPring-8 and BL5 and NW-12 of KEK for technical help during data collection, and A. Hamada for technical assistance. This work was supported by grants from JSPS for young scientists, MEXT for priority areas of science, the Mitsubishi Foundation, the Sumitomo Foundation and AIST to K.T., as well as by a PRESTO and SORST Program grant from JST (Japan Science and Technology) and a grant from MEXT to O.N. Author Contributions K.T. carried out structural determination and mutant analysis, and wrote the paper with editing from R.I. and O.N. R.I., S.F. and O.N. assisted with the structural determination. All authors discussed the results and commented on the manuscript. O.N. supervised the work.

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Affiliations

  1. Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Higashi, Tsukuba-shi, Ibaragi 305-8565, Japan

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

    • Ryuichiro Ishitani
    • , Shuya Fukai
    •  & Osamu Nureki
  3. SORST, JST, Honcho, Kawaguchi-shi, Saitama 332-0012, Japan

    • Osamu Nureki

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Competing interests

Coordinates and structure factors have been deposited in the Protein Data Bank, under the accession codes 2DR5, 2DR7, 2DR8, 2DR9, 2DRA, 2DRB and 2DVI for the mini-D stage, the mini-DC stage, the mini-DC + CTP stage, the mini-DCC stage, the mini-DCC + ATP stage, the mini-DCCA stage and the mini-DCC + CTP misrecognition complex, respectively. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Corresponding author

Correspondence to Osamu Nureki.

Supplementary information

PDF files

  1. 1.

    Supplementary Figures

    This file contains Supplementary Figures 1–8.

Videos

  1. 1.

    Supplementary Movie 1

    This movie shows the CCA-adding dynamics of the CCA-adding enzyme, primer tRNA and incoming NTP, highlighting the ‘open’ to ‘closed’ conformational transition of the enzyme

  2. 2.

    Supplementary Movie 2

    This movie shows the close-up view of the CCA-adding dynamics in the catalytic site.

Word documents

  1. 1.

    Supplementary Notes

    This file contains Supplementary Discussions, Supplementary Methods, Supplementary Table, Supplementary Movie Legends and additional references.

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DOI

https://doi.org/10.1038/nature05204

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