On the Rule of Different Subunits ofDNA-Dependent RNA Polymerase from E. coli in the Transcription Process

DNA-dependent RNA polymerase from E. coli is now routinely obtained in our laboratory in a highly purified form (more than 95 % pure) in large quantities (up to 300 mg/kg cell wet weight) by two alternative procedures. The first one involves the use of polymin P for the precipitation ofthe enzyme from the crude extract, DEAE chromatography, ammonium sulfate precipitation, and two zonal centrifugation steps performed at different ionic strength for the isolation of a full enzyme which contains about 50 % of the stoichiometrically saturating amount of initiation factor a (Zillig et el., 1970). The second procedure involves phos-phorylcellulose (PC) chromatography (Burgess et al., 1969) following the DEAE step of the first procedure, which yields minimal or core enzyme. From the flow-through of the PC column, another DEAE step and sucrose gradient sedimentation yield purified initiation factor a. The enzyme has been dissociated into its sub-units fl', fl, 2 • 0r and a by 6 M urea at pH 9 (Zillig et al., 1970), (Fig. 1). Preparative separation FIGURE 1. Subunit patterns of DNA-dependent RNA polymerase from E. coll. left: polyacrylamide gel electrophoresis in the presence of 6 ~ urea and 0.1 % SDS at pH 9, 7.5 % gel. right: eleetrophoresis on cellulose acetate sheet (cellogel