The Escherichia coli RlmN methyltransferase is a dual-specificity enzyme that modifies both rRNA and tRNA and controls translational accuracy

RlmN methylates tRNA^Chimera_UUG both in vivo and in vitro. (A) The sequence and secondary structure of the scaffold tRNA is shown together with a representative section of its HPLC chromatogram (D and Ψ modifications elute at early times and are not included in the figure). (B) The tRNA^Chimera_UUG sequence (red nucleotides indicate the ASL from tRNA^Gln_cmnm5s2UUG) is shown together with a section of the corresponding chromatogram. (C) Synthesis in vivo of m²A on tRNA^Chimera_UUG (left panel) is prevented by mutation A37C (middle panel) or by the expression of the wild-type tRNA^Chimera_UUG in ΔrlmN cells (right panel). (D) In vitro methylation of tRNA^Chimera_UUG purified from ΔrlmN cells is carried out by in vitro-reconstituted RlmN in a SAM-dependent manner (red lines). Reconstituted RlmN does not work on in vitro synthesized tRNA^Gln_UUG (black lines). Solid and discontinuous lines indicate that the modification reactions were performed in the presence and absence of SAM, respectively. Absorbance was monitored at 260 nm. (mAU) Absorbance units ×10⁻³.