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

Glycerol gradient sedimentation. Zonal velocity sedimentation was performed as described in the Materials and Methods. Aliquots (15 μL) of even-numbered glycerol gradient fractions of CthHen1 cosedimented with marker proteins (A), or by itself (B) were analyzed by SDS-PAGE. The Coomassie-blue stained gels are shown. The CthHen1, catalase, BSA, and cytochrome c polypeptides are indicated in A. Molecular weight calibrations for the PAGE analysis (kilodalton) are indicated on the left. The methyltransferase activity profile for the glycerol gradient in B is shown in C. Reaction mixtures (10 μL) containing 25 mM Tris-HCl (pH 8.5), 1 mM MnCl₂, 20 μM [³H-CH₃]AdoMet, 10 μM 24-mer RNA, and 2 μL of the indicated gradient fraction were incubated for 60 min at 45°C. The extents of ³H-methyl transfer to RNA are plotted.