Aquifex aeolicus tRNA (N2,N2-Guanine)-dimethyltransferase (Trm1) Catalyzes Transfer of Methyl Groups Not Only to Guanine 26 but Also to Guanine 27 in tRNA*

  1. Takako Awai‡,
  2. Satoshi Kimura§,
  3. Chie Tomikawa‡,
  4. Anna Ochi‡,
  5. Ihsanawati,
  6. Yoshitaka Bessho‖,
  7. Shigeyuki Yokoyama‖**,
  8. Satoshi Ohno‡‡,
  9. Kazuya Nishikawa‡‡,
  10. Takashi Yokogawa‡‡,
  11. Tsutomu Suzuki§ and
  12. Hiroyuki Horiत,1
  1. From the ‡Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, Bunkyo 3, Matsuyama, Ehime 790-8577,
  2. the §Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8656,
  3. the Systems and Structural Biology Center, Yokohama Institute, RIKEN, Suehiro-cho 1-7-22, Tsurumi-ku, Yokohama, Kanagawa 230-0045,
  4. the ‖RIKEN SPring-8 Center, Harima Institute, Kouto 1-1-1, Sayo, Hyogo 679-5148,
  5. the **Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033,
  6. the ‡‡Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido 1-1, Gifu, Gifu 501-1193, and
  7. the §§Venture Business Laboratory, Ehime University, Bunkyo 3, Matsuyama, Ehime 790-8577, Japan
  1. 1 To whom correspondence should be addressed. Tel.: 81-89-927-8548; Fax: 81-89-927-9941; E-mail: hori{at}eng.ehime-u.ac.jp.

Abstract

Transfer RNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes N2,N2-dimethylguanine formation at position 26 (m22G26) in tRNA. In the reaction, N2-guanine at position 26 (m2G26) is generated as an intermediate. The trm1 genes are found only in archaea and eukaryotes, although it has been reported that Aquifex aeolicus, a hyper-thermophilic eubacterium, has a putative trm1 gene. To confirm whether A. aeolicus Trm1 has tRNA methyltransferase activity, we purified recombinant Trm1 protein. In vitro methyl transfer assay revealed that the protein has a strong tRNA methyltransferase activity. We confirmed that this gene product is expressed in living A. aeolicus cells and that the enzymatic activity exists in cell extract. By preparing 22 tRNA transcripts and testing their methyl group acceptance activities, it was demonstrated that this Trm1 protein has a novel tRNA specificity. Mass spectrometry analysis revealed that it catalyzes methyl transfers not only to G26 but also to G27 in substrate tRNA. Furthermore, it was confirmed that native tRNACys has an m22G26m2G27 or m22G26m22G27 sequence, demonstrating that these modifications occur in living cells. Kinetic studies reveal that the m2G26 formation is faster than the m2G27 formation and that disruption of the G27-C43 base pair accelerates velocity of the G27 modification. Moreover, we prepared an additional 22 mutant tRNA transcripts and clarified that the recognition sites exist in the T-arm structure. This long distance recognition results in multisite recognition by the enzyme.

Footnotes

  • ↵3 T. Yokogawa, S. Ohno, and K. Nishikawa, manuscript in preparation.

  • ↵* This work was supported in part by Japan Society for the Promotion of Science Research Fellowship for Young Scientists 20-4827 (to C. T.), Grant-in-aid for Science Research on Priority Areas 20034041 (to H. H.), and Grant-in-aid for Science Research 19350087 (to H. H.) from the Ministry of Education, Science, Sports, and Culture of Japan.

  • ↵Graphic The on-line version of this article (available at http://www.jbc.org) contains supplemental Experimental Procedures and Table 1.

  • ↵2 The abbreviations used are:

    AdoMet

    S-adenosyl-l-methionine

    LC/MS

    liquid chromatography/mass spectrometry

    MS/MS

    tandem mass spectrometry.

    • Received March 18, 2009.
    • Revision received May 13, 2009.
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  1. First Published on June 2, 2009 doi: 10.1074/jbc.M109.020024 The Journal of Biological Chemistry 284, 20467-20478.
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