Identification of a novel tRNA wobble uridine modifying activity in the biosynthesis of 5-methoxyuridine

Derivatives of 5-hydroxyuridine (ho⁵U), such as 5-methoxyuridine (mo⁵U) and 5-oxyacetyluridine (cmo⁵U), are ubiquitous modifications of the wobble position of bacterial tRNA that are believed to enhance translational fidelity by the ribosome. In gram-negative bacteria, the last step in the biosynthesis of cmo⁵U from ho⁵U involves the unique metabolite car-boxy S-adenosylmethionine (Cx-SAM) and the carboxymethyl transferase CmoB. However, the equivalent position in the tRNA of Gram-positive bacteria is instead mo⁵U, where the methyl group is derived from SAM and installed by an unknown methyltransferase. By utilizing a cmoB-deficient strain of Escherichia coli as a host and assaying for the formation of mo⁵U in total RNA isolates with methyltransferases of unknown function from Bacillus subtilis, we found that this modification is installed by the enzyme TrmR (formerly known as YrrM). Furthermore, X-ray crystal structures of TrmR with and without the anticodon stemloop of tRNA^Ala have been determined, which provide insight into both sequence and structure specificity in the interactions of TrmR with tRNA.