RNA polymerase from Bacillus subtilis: isolation of core and holo enzyme by DNA-cellulose chromatography.
A new procedure for the purification of B. subtilis RNA polymerase, based on mild lysis of cells, low speed centrifugation, gel filtration, DEAE-Sephadex chromatography and affinity chromatography on DNA-cellulose, yields three forms of enzyme referred here as enzyme A, B and C. As revealed by SDS gel electrophoresis, enzyme A has the subunit structure of core polymerase plus some small polypeptides. Its catalytic properties are similar to those of core polymerase. Enzyme B has the composition of core polymerase. Both enzymes A and B can be stimulated by the addition of beta factor. Enzyme C has the holo-enzyme composition. The pattern of sensitivity of the three forms of enzyme towards KCl are very different: enzymes A and B, even at low concentration of salt, are inhibited with all the DNA templates tested, whereas enzyme C shows a pattern of stimulation specific for each DNA tested. The transcripts of the three enzymes on phage SPP1 DNA template have been analyzed by hybridization to the separated strands. Only enzyme C selectively transcribed the H strands.
D001412:Bacillus subtilis / Q000201:enzymology*; D001435:Bacteriophages; D002846:Chromatography, Affinity; D004269:DNA, Bacterial; D004279:DNA, Viral; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D046911:Macromolecular Substances; D009693:Nucleic Acid Hybridization; D011189:Potassium Chloride / Q000494:pharmacology; D012313:RNA / Q000096:biosynthesis; D013698:Templates, Genetic; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D004279:DNA, Viral; D046911:Macromolecular Substances; D012313:RNA; D011189:Potassium Chloride; D012321:DNA-Directed RNA Polymerases