Folate-/FAD-dependent tRNA methyltransferase from Thermus thermophilus regulates other modifications in tRNA at low temperatures.
RNAs contain structurally and functionally important modified nucleosides. Methylation, the most frequent RNA modification in all living organisms, mostly relies on SAM (S-adenosylmethionine)-dependent methyltransferases. TrmFO was recently discovered as a unique tRNA methyltransferase using instead methylenetetrahydrofolate and reduced flavin adenine dinucleotide (FAD) as essential cofactors, but its mechanism has remained elusive. Here, we report that TrmFO carries an active tRNA-methylating agent and characterize it as an original enzyme-methylene-FAD covalent adduct by mass spectrometry and a combination of spectroscopic and biochemical methods. Our data support a novel tRNA methylating mechanism.