Bacterial Hen1 is a 3′ terminal RNA ribose 2′-O-methyltransferase component of a bacterial RNA repair cassette

Product analysis and substrate specificity. (A,B) Reaction mixtures (30 μL) containing 25 mM Tris-HCl (pH 8.5), 0.5 mM MnCl₂, 20 μM [³H-CH₃]AdoMet, 8 μM CthHen1 (A) or 8 μM CthHen1(259–465) (B), and 40 μM 24-mer RNA (where indicated by +) were incubated for 30 min at 45°C and then quenched with EDTA. The RNA-containing mixtures were split into three aliquots. One was stored at −20°C, while the other two were adjusted to either 0.3 M KOH or 0.3 M KCl and incubated for 15 h at 37°C. The KOH samples were then neutralized by adding HCl to 0.3 M final concentration. The samples were analyzed by PEI cellulose TLC in 50 mM ammonium sulfate. Unlabeled marker nucleotides analyzed in parallel were detected by UV autofluorescence. The ³H-labeled material was visualized by autoradiography. (C) Methyl acceptor specificity. Reaction mixtures (10 μL) containing 25 mM Tris-HCl (pH 8.5), 0.5 mM MnCl₂, 20 μM [³H-CH₃]AdoMet, 8 μM CthHen1, and either no added nucleic acid (none) or 10 μM 24-mer RNA with a 3′ terminal ribose (2′-OH), a 3′-terminal deoxyribose (2′-H), or a 3′-terminal 2′-O-methylribose (2′-OMe), were incubated for 30 min at 45°C. The extents of ³H-methyl transfer to the TLC origin are plotted. (D) Product inhibition. Reaction mixtures (10 μL) containing 25 mM Tris-HCl (pH 8.5), 0.5 mM MnCl₂, 20 μM [³H-CH₃]AdoMet, 10 μM 24-mer RNA, 4 μM CthHen1, and 0, 10, 20, 40 or 80 μM AdoHcy were incubated for 30 min at 45°C. The extents of ³H-methyl transfer to RNA are plotted as a function of AdoHcy concentration.