Modified nucleosides m3Ψ and m3U formed by RlmH in vivo and in vitro. HPLC analysis of nucleoside composition of a 23S rRNA fragment comprising nucleotides 1777–1922. The 23S rRNA fragments were isolated by RNase H treatment and gel electrophoresis from 70S ribosomes of E. coli wild-type strain (MG1655), isogenic strain lacking rlmH (ΔrlmH), or both rlmH and rluD genes (ΔrlmH/ΔrluD). For in vivo experiments, strains were complemented with a plasmid (pBAD-rlmH) overexpressing RlmH protein and for in vitro experiments, purified 70S ribosomes were treated with RlmH protein in vitro. Peaks corresponding to four standard nucleosides (C, U, G, and A) and three modified nucleosides; 3-methylpseudouridine (m3Ψ), 3-methyluridine (m3U), and 2-methylguanosine (m2G) are indicated. Nonspecific peak resulting from experimental conditions is marked with an X.
