Substrate tRNA Recognition Mechanism of Eubacterial tRNA (m1A58) Methyltransferase (TrmI)

• Published in: The Journal of biological chemistry 2015-02, Vol.290 (9), p.5912-5925
• Main Authors: Takuma, Hiroyuki, Ushio, Natsumi, Minoji, Masayuki, Kazayama, Ai, Shigi, Naoki, Hirata, Akira, Tomikawa, Chie, Ochi, Anna, Hori, Hiroyuki
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites - genetics; Electrophoresis, Polyacrylamide Gel; Kinetics; Methylation; Models, Molecular; Mutation; Nucleic Acid Conformation; Protein Binding; Protein Structure, Tertiary; Protein Synthesis and Degradation; RNA Methylation; RNA Methyltransferase; RNA Modification; RNA, Bacterial - chemistry; RNA, Bacterial - genetics; RNA, Bacterial - metabolism; RNA, Transfer - chemistry; RNA, Transfer - genetics; RNA, Transfer - metabolism; RNA, Transfer, Phe - chemistry; RNA, Transfer, Phe - genetics; RNA, Transfer, Phe - metabolism; RNA, Transfer, Thr - chemistry; RNA, Transfer, Thr - genetics; RNA, Transfer, Thr - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Substrate Specificity; Thermophile; Thermus thermophilus - enzymology; Thermus thermophilus - genetics; Thermus thermophilus - metabolism; Transfer RNA (tRNA); tRNA Methyltransferases - chemistry; tRNA Methyltransferases - genetics; tRNA Methyltransferases - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.606038

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 10 deletion tRNA variants: TrmI methylated transcripts containing the aminoacyl stem, variable region, and T-arm. The 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 tRNAGGUThrGGUThr contains C60 instead of U60. The tRNAGGUThr 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 tRNAGGUThr had a low affinity for TrmI than tRNAPhe. Furthermore, analysis of tRNAGGUThr 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 tRNAGGUThr from the wild-type strain indicated that less than 50% of tRNAGGUThr contained m1A58. Thus, the results from the in vitro experiments were confirmed by the in vivo methylation patterns. tRNA methyltransferases specifically recognize substrate tRNAs. To clarify the tRNA recognition mechanism of TrmI, three tRNA species and 45 variants were analyzed in vitro and in vivo. TrmI recognizes the aminoacyl stem, variable region, C56, purine 57, A58, and U60 in the T-loop of tRNA. Our in vitro experimental results explain the regulation of in vivo methylation levels in tRNAs.