RlmN modifies both rRNA and tRNA. (A) rRNA/tRNA HPLC-nucleoside profile of the Escherichia coli ΔrlmN mutant. After isolation and P1 digestion, bulk rRNA (left) and tRNA (right) from the wild-type and ΔrlmN strains were separately analyzed by HPLC. Nucleoside m2A was identified by its elution time and UV spectra. Note that nucleoside t6A (present at position 37 of some tRNAs that are not RlmN substrates) is unaffected in the ΔrlmN cells. (B) In vivo complementation of the ΔrlmN mutant. Recombinant protein His-RlmN, expressed from pET15b-his-rlmN, restores the m2A synthesis in ΔrlmN cells (right panel). Wild-type and ΔrlmN cells containing pET15b (left and middle panels, respectively) were used as controls. Absorbance was monitored at 260 nm. (mAU) Absorbance units ×10−3.
