Partial purification and characterization of RNase P from Dictyostelium discoideum

Ribonuclease P (RNase P) from Dictyostelium discoideum has been purified 470-fold. D. discoideum RNase P cleaves the precursor to Schizosaccharomyces pombe suppressor tRNA(Ser) at the same site as S. pombe RNase P, producing the mature 5' end of tRNA(Ser). pH and temperature optima for enzyme activity are 7.6 and 37 degrees C, respectively. The enzyme shows optimal activity in the presence of 5 mM MgCl2 and 10 mM NH4Cl or 5 mM KCl. The apparent Km for the S. pombe tRNA precursor derived from the supS1 tRNA(Ser) gene is 240 nM, and the apparent Vmax is 3.6 pmol/min. Inhibition of D. discoideum RNase P by proteinase K and micrococcal nuclease strongly indicates that the activity requires both protein and RNA components. In cesium sulfate density gradients, the enzyme has a buoyant density of 1.23 g/ml, indicating a low RNA/protein ratio for the holoenzyme.