Transcription of Isolated Nuclei and Nucleoli by Exogenous RNA Polymerases A and B

Abstract

Both forms A and B RNA polymerases solubilised from rat liver nuclei transcribed templates within these organelles when added exogenously to freshly prepared nuclei. The enzymes initiated more efficiently in the presence of KCL than ammonium sulphate and required manganese rather than magnesium as the divalent cation. Form A enzyme initiated most successfully at 375 mM KC6, activity was proportional to the amount of template added and continued linearly for at least 30 min. Form B enzyme initiated with two ionic strength optima, 125 mM and 500 mM KCl. Activity in the latter case was critically dependent on the enzyme: nuclei ratio. In both instances incorporation of nucleotide precurors was linear for less than 20 min. Form A enzyme synthesised products with a size distribution mainly larger than 18 S; form B enzyme synthesised products of mainly less than 5 S at 125 mM KCl and about 10 S at 500 mM KCl. Subfractionation of nuclei indicated that exogenous RNA polymerase A activity and form B at 125 mM KCl were occurring in nucleoli; form B activity at 500 mM KCl was nucleoplasmic. Measurements of U : G ratios in the RNA products suggested that exogenous form A was synthesising species with similar base ratios to the ribosomal RNA precurosrs. Both enzymes formed rifamycin AF/0-13 resistant complexes with nucleolar templates. Size analyses of products showed that whereas form B enzyme synthesised very small RNA species, RNA polymerase A produced a range of species of similar sizes to the ribosomal RNA precurosors.