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April 4th, 2025
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Ehime University
biochemistry
biorxiv

A transfer RNA methyltransferase with an unusual domain composition catalyzes 2'-O-methylation at position 6 in tRNA.

Matsuda, T.Open in Google Scholar•Yamagami, R.Open in Google Scholar•Ihara, A.Open in Google Scholar•Suzuki, T.Open in Google Scholar•Hirata, A.Open in Google Scholar•Hori, H.Open in Google Scholar

Thermococcus kodakarensis tRNATrp contains 2\'-O-methylcytidine at position 6 (Cm6). However, the tRNA methyltransferase responsible for the modification has not been identified. Using comparative genomics we predicted TK1257 as a candidate for the modification. Biochemical and mass spectrometry studies of purified recombinant TK1257 gene product show it to possess a tRNA methyltransferase activity for Cm6 formation. This protein has a highly unusual composition of domains, containing N-terminal ferredoxin-like, SPOUT catalytic and THUMP domains. Previous to this study, all known THUMP-related tRNA methyltransferases were shown to contain a Rossmann fold catalytic domain and the nucleosides they produced were N2-methylguanosine and/or N2, N 2-dimethylguanosine. Therefore, our findings extend the knowledge of architecture of tRNA methyltransferases. We named the TK1257 gene product TrmTS and showed it can synthesize Am6 and Um6 as well as Cm6. A trmTS gene deletion strain showed slight growth retardation at high temperatures. Site-directed mutagenesis studies based on structural model revealed catalytically and structurally important amino acid residues in TrmTS and identified a TrmTS-specific linker is structurally essential. We showed that TrmTS recognizes the 3\'-CCA terminal region and a stretch loop connected with at least two stems in RNA. Finally, we constructed a model of the binding between TrmTS and tRNA.

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