Determinants of tRNA Recognition by the Radical SAM Enzyme RlmN

RlmN, a bacterial radical SAM methylating enzyme, has the unusual ability to modify two distinct types of RNA: 23S rRNA and tRNA. In rRNA, RlmN installs a methyl group at the C2 position of A2503 of 23S rRNA, while in tRNA the modification occurs at nucleotide A37, immediately adjacent to the anticodon triplet. Intriguingly, only a subset of tRNAs that contain an adenosine at position 37 are substrates for RlmN, suggesting that the enzyme carefully probes the highly conserved tRNA fold and sequence features to identify its targets. Over the past several years, multiple studies have addressed rRNA modification by RlmN, while relatively few investigations have focused on the ability of this enzyme to modify tRNAs. In this study, we utilized in vitro transcribed tRNAs as model substrates to interrogate RNA recognition by RlmN. Using chimeras and point mutations, we probed how the structure and sequence of RNA influences methylation, identifying position 38 of tRNAs as a critical determinant of substrate recognition. We further demonstrate that, analogous to previous mechanistic studies with fragments of 23S rRNA, tRNA methylation requirements are consistent with radical SAM reactivity. Together, our findings provide detailed insight into tRNA recognition by a radical SAM methylating enzyme.