Metal specificity of PhoRtcB. (A) Reaction mixtures (10 µL) containing 50 mM Tris-HCl (pH 8.0), 10 µM [α32P]GTP, 5 µM RtcB, and 2 mM of the indicated divalent cation (as the chloride salt) were incubated at 75°C for 20 min. Divalent cation was omitted from an RtcB-containing control reaction (lane –). (B) Reaction mixtures (10 µL) containing 50 mM Tris-HCl (pH 8.0), 10 µM [α32P]GTP, 5 µM RtcB, 1 mM MnCl2, and 1 mM of the indicated divalent cation were incubated at 75°C for 20 min. (C,D) Reaction mixtures (10 µL) containing 50 mM Tris-HCl (pH 8.0), 10 µM [α32P]GTP, 5 µM RtcB, 2 mM MnCl2, and increasing concentrations of ZnCl2 or CuCl2 (as specified above the lanes) were incubated at 75°C for 20 min. The reaction products were analyzed by SDS-PAGE. Autoradiographs of the gels are shown. The positions and sizes of prestained marker proteins are indicated on the left. The relative extents of guanylylation in these assays are specified below the lanes, normalized to those of the manganese-only reactions (defined as 100%).
