DNA-dependent RNA polymerase from Pseudomonas BAL-31. I. Purification and properties of the enzyme.

Abstract

The DNA-dependent RNA polymerase from Pseudomonas BAL-31, the host for bacteriophage PM2, has been purified 154-fold using differential centrifugation, chromatography on DEAE-cellulose, ammonium sulfate precipitation, and sucrose gradient centrifugations at low and high ionic strength. The resulting enzyme is free of enzyme activities which could interfere with transcription studies and is greater than 85% pure as judged by polyacrylamide gel electrophoresis. Like other bacterial RNA polymerases, its subunit structure is beta'beta sigma alpha2. From gel electrophoresis the beta', beta, and alpha subunits have approximately the same molecular weights as those from Escherichia coli, whereas the sigma subunit is 5% larger (89,000 vs. 85,000). A summation of the subunits yields a molecular weight of 485,000 for the holoenzyme. Like other bacterial RNA polymerases, it sediments as a monomer (15 S) at low ionic strength (0.065) and as a dimer (22 S) at high ionic strength (0.75). Its activity is stimulated three-fold by monovalent cations (K+,NH4+, NA+) with additional stimulation provided by divalent cations (Mg2plus, Mn2plus). The transcription of phage PM2 form I (supercoiled) DNA has an ionic strength optimum of 0.26 for continuous long-term synthesis, and over an ionic strength range of 0.09-0.46 "plateau-type" kinetics are not observed. The sigma subunit is required for optimal PM2 transcription. The enzyme is sensitive to the same inhibitors of transcription as the RNA polymerase from E. coli, it has a temperature optimum of 28 degrees, and it is 50% inactivated by heating 10 min at 41 degrees. It has template preference similar to E. coli polymerase and shows little preference for homologous templates. With various DNAs the order of template activities is T7 greater than PM2 I congruent to T4 greater than PM2 II (relaxed circular form) greater than lambda-c greater than calf thymus greater than BAL-31 DNA. Phage PM2 form I DNA is transcribed at a twofold greater rate than PM2 form II DNA by this enzyme.