Characterization of a heat-stable enzyme possessing GTP-dependent RNA ligase activity from a hyperthermophilic archaeon, Pyrococcus furiosus

  1. Akio Kanai12,
  2. Asako Sato1,
  3. Yoko Fukuda1,
  4. Kiyoshi Okada3,
  5. Takashi Matsuda3,
  6. Taiichi Sakamoto34,
  7. Yutaka Muto4,
  8. Shigeyuki Yokoyama45,
  9. Gota Kawai34 and
  10. Masaru Tomita12
  1. 1Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan
  2. 2Systems Biology Program, Graduate School of Media and Governance, Keio University, Fujisawa 252-8520, Japan
  3. 3Department of Life and Environmental Sciences, Faculty of Engineering, Chiba Institute of Technology, Narashino-shi, Chiba 275-0016, Japan
  4. 4RIKEN, Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan
  5. 5Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Abstract

Using an expression protein library of a hyperthermophilic archaeon, Pyrococcus furiosus, we identified a gene (PF0027) that encodes a protein with heat-stable cyclic nucleotide phosphodiesterase (CPDase) activity. The PF0027 gene encoded a 21-kDa protein and an amino acid sequence that showed ∼27% identity to that of the 2′-5′ tRNA ligase protein, ligT (20 kDa), from Escherichia coli. We found that the purified PF0027 protein possessed GTP-dependent RNA ligase activity and that synthetic tRNA halves bearing 2′,3′-cyclic phosphate and 5′-OH termini were substrates for the ligation reaction in vitro. GTP hydrolysis was not required for the reaction, and GTPγS enhanced the tRNA ligation activity of PF0027 protein, suggesting that the ligation step is regulated by a novel mechanism. In comparison to the strong CPDase activity of the PF0027 protein, the RNA ligase activity itself was quite weak, and the ligation product was unstable during in vitro reaction. Finally, we used NMR to determine the solution structure of the PF0027 protein and discuss the implications of our results in understanding the role of the PF0027 protein.

Keywords:

Keywords

Footnotes

  • Reprint requests to: Akio Kanai, Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan; e-mail: akio{at}sfc.keio.ac.jp; fax: 81-235-29-0525.

  • Abbreviations: CPDase, cyclic nucleotide phosphodiesterase; NCBI, National Center for Biotechnology Information; PDB, protein data bank; FAM, 6-carboxyfluorescein; CE-TOFMS, capillary electrophoresis-time of flight mass spectrometry; HSQC, heteronuclear single quantum coherence spectroscopy; DQF-COSY, double quantum filtered-correlation spectroscopy; TOCSY, total correlation spectroscopy; HP-COSY, proton phosphorus-correlation spectroscopy.

  • Article and publication are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1122109.

    • Received April 4, 2008.
    • Accepted November 24, 2008.

This Article

  1. Published in Advance January 20, 2009, doi: 10.1261/rna.1122109 RNA Copyright © 2009 RNA Society

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