Purification and DNA‐Binding Properties of RNA Polymerase from Bacillus subtilis

Abstract

Four RNA-polymerizing activities having different subunit composition can be purified from uninfected and from [phage] SPO1-infected B. subtilis. Lysozyme and sodium deoxycholate are used for lysing the cells. Polymin P is used for precipitating nucleic acids and DEAE-cellulose chromatography allows separation of enzymatic activity from the residual Polymin P. After these common steps, one can purify core + .sigma. + .delta. by chromatography on single-stranded DNA-agarose followed by gel filtration while pure core + .sigma. can be obtained by chromatography on double-stranded DNA cellulose. Core + .delta. is obtained by high-salt sucrose/glycerol gradient centrifugation. The host enzyme modified by the product of gene 28 of phage SPO1 can be purified from SPO1 infected cells by chromatography on DNA cellulose (or DNA agarose) followed by chromatography on phosphocellulose. The pH and salt dependance of the initial rate of RNA synthesis of core + .sigma. was investigated using SPO1 and SPP1 DNA as templates. The optimum pH for the initial rate of transcription is 8.2 at 30.degree. C in 50 mM N,N-bis(2-hydroxyethyl)glycine buffer, and the optimum Na+ concentration is between 0.1-0.15 M. The kinetics of formation and of dissociation of non-filterable complexes between SPP1 DNA and core + .sigma. have been analyzed at different cationic concentrations. The value of the rate constant of dissociation in 0.1 M NaCl at 30.degree. C is kd = 2.16 .times. 10-4 s-1. The value of the rate constant of association, under the same conditions, is ka = 5.5 .times. 108 M-1 s-1; this value is compatible with a diffusion-controlled reaction for promoter selection.