In vitro Synthesis of Ribosomal RNA by Bacillus subtilis RNA Polymerase

• Published in: Proceedings of the National Academy of Sciences - PNAS 1972-02, Vol.69 (2), p.407-411
• Main Authors: Hussey, Caroline, Pero, Janice, Shorenstein, Rosalind G., Losick, Richard
• Format: Article
• Language: English
• Subjects: Bacillus subtilis - enzymology; Biological Sciences: Biochemistry; Chromatography; Chromatography, DEAE-Cellulose; DNA; DNA, Bacterial - metabolism; Electrophoresis, Disc; Enzymes; Genes; Liquids; Mutation; Nucleic Acid Denaturation; Nucleic Acid Hybridization; Phosphorus Isotopes; Protein Binding; Ribosomal RNA; RNA; RNA Nucleotidyltransferases - isolation & purification; RNA Nucleotidyltransferases - metabolism; RNA, Ribosomal - biosynthesis; rRNA genes; Sporulation; Steepest descent method; Templates, Genetic; Tritium
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.69.2.407

Two kinds of hybridization competition experiments show that Bacillus subtilis RNA polymerase synthesizes ribosomal RNA (rRNA) in vitro with B. subtilis DNA as a template. First, RNA synthesized in vitro competes with the hybridization of [32P]rRNA synthesized in vivo to the heavy strand of B. subtilis DNA. Second, unlabeled rRNA synthesized in vivo competes with the hybridization of [3H]RNA synthesized in vitro to denatured DNA or heavy-strand DNA, but not to light-strand DNA. The ability of RNA polymerase holoenzyme to synthesize rRNA in vitro is not lost after extensive purification. RNA polymerase core enzyme, however, which is missing the σ factor, synthesizes little rRNA in vitro. RNA polymerase purified from wild-type sporulating cells synthesizes little rRNA in vitro, while the in vitro synthesis of rRNA by RNA polymerase from stationary phase cells of the sporulation-defective mutant rfr 10 is apparently unimpaired.