Substrate tRNA Recognition Mechanism of Eubacterial tRNA (m1A58) Methyltransferase (TrmI) [RNA]

January 15th, 2015 by Takuma, H., Ushio, N., Minoji, M., Kazayama, A., Shigi, N., Hirata, A., Tomikawa, C., Ochi, A., Hori, H.

TrmI generates N1-methyladenosine at position 58 (m1A58) in tRNA. The Thermus thermophilus tRNAPhe transcript was methylated efficiently by T. thermophilus TrmI, whereas the yeast tRNAPhe transcript was poorly methylated. Fourteen chimeric tRNA transcripts derived from these two tRNAs revealed that TrmI recognized the combination of aminoacyl stem, variable region and T-loop. This was confirmed by ten deletion tRNA variants: TrmI methylate transcripts containing the aminoacyl stem, variable region and T-arm. Requirement for the T-stem itself was confirmed by disrupting the T-stem. Disrupting the interaction between T- and D-arms accelerated the methylation, suggesting that this disruption is included in part of the reaction. Experiments with 17 point mutant transcripts elucidated the positive sequence determinants, C56, purine 57, A58, and U60. Replacing A58 with inosine and 2-aminopurine completely abrogated methylation, demonstrating that the 6-amino group in A58 is recognized by TrmI. T. thermophilus tRNAThrGGU contains C60 instead of U60. The tRNAThrGGU transcript was poorly methylated by TrmI and replacing C60 with U increased the methylation, consistent with the point mutation experiments. A gel shift assay revealed that tRNAThrGGU had a low affinity for TrmI than tRNAPhe. Furthermore, analysis of tRNAThrGGU purified from the trmI gene disruptant strain revealed that the other modifications in tRNA accelerated the formation of m1A58 by TrmI. Moreover, nucleoside analysis of tRNAThrGGU from the wild-type strain indicated that less than 50% of tRNAThrGGU contained m1A58. Thus, the results from the in vitro experiments were confirmed by the in vivo methylation patterns.