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¹ Department of Applied Chemistry, Faculty of Engineering, Ehime University, Bunkyo 3, Matsuyama 790-8577, Japan
² Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8656, Japan
³ Department of Molecular Biology, School of Life Science, Tokyo University of Pharmacy and Life Science, Horinouchi 1432-1, Hachioji 192-0392, Japan
⁴ Venture Business Laboratory, Ehime University, Bunkyo 3, Matsuyama 790-8577, Japan
⁵ Cell-free Science and Technology Research Center, Ehime University, Matsuyama, 790-8577, Japan

Transfer RNA (m¹G37) methyltransferase (TrmD) catalyzes methyl-transfer from S-adenosyl-L-methionine to the N¹ atom of G37 in tRNA. In Escherichia coli cells, TrmD methylates tRNA species possessing a G36G37 sequence. It was previously believed that G36 was the positive determinant of TrmD recognition. In the current study, we demonstrate that TrmD from Aquifex aeolicus methylates tRNA transcripts possessing an A36G37 sequence as well as tRNA transcripts possessing a G36G37 sequence. In contrast, tRNA transcripts possessing pyrimidine36G37 were not methylated at all. These substrate specificities were confirmed by an in vitro kinetic assay using 16 tRNA transcripts. The modified nucleoside and the position in yeast tRNA^Phe transcript were confirmed by LC/MS. Furthermore, nine truncated tRNA molecules were tested to clarify the additional recognition site. Unexpectedly, A. aeolicus TrmD protein efficiently methylated the micro helix corresponding to the anti-codon arm. Because the disruption of the anti-codon stem caused the complete loss of the methyl group acceptance activity, the anti-codon stem is essential for the recognition. Moreover, the existence of the D-arm structure inhibited the activity. Recently, it was reported that E. coli TrmD methylates yeast tRNA^Phe harboring a sequence A36G37. Thus, recognition of the purine36G37 sequence is probably common to eubacteria TrmD proteins.

^aPresent address: Institute of Environmental Microbiology, Kyowa Kako Co. Ltd, Tadao 2-15-5, Machida 194-0035, Japan

^* Correspondence: E-mail: hori{at}eng.ehime-u.ac.jp

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