Purification and Properties of Two RNA Polymerases from Sporulating Cells of Bacillus subtilis

DNA-dependent RNA polymerase (EC 2.7.7.6) was purified from sporulating cells of Bacillus subtilis 168. The elution pattern of the enzyme from a DNA-cellulose column revealed that sporulating cells at t3.5 contained two forms of RNA polymerase, holoenzyme (ββ′α2σ) and δ1 enzyme (ββ′α2δ1) in addition to the core enzyme (ββ′α2). The molar ratio of δ1 subunit to core enzyme was 1.00 throughout the δ1 enzyme fraction and the molecular weight of the δ1 factor was 29000. The optimum concentration of metal ions for transcription was different for each enzyme when various DNAs were used as template. KCl had no effect on the activity of δ1 enzyme until 0.3 M, although the addition of more than 0.15 M KCl decreased the activity of holoenzyme with B. subtilis DNA as template. Holoenzyme was stable at high KCl concentrations but δ1 enzyme was unstable under such conditions. Although both enzyme forms had almost the same specific activity on phage DNAs, δ1 enzyme had a higher specific activity on poly[d(A-T)] and B. subtilis DNA than holoenzyme. Vegetative σ factor stimulated the activity of sporulation core enzyme with poly[d(A-T)] and φe DNA as templates, but the σ factor did not stimulate the activity of the δ1 enzyme with poly[d(A-T)] and φ29 DNA as templates; however, σ factor did stimulate δ1 enzyme activity twofold with φe DNA as template. At a concentration of 1 μg/ml the polypeptide antibiotic, netropsin, inhibited sporulation and the synthesis of the δ1 enzyme almost completely. Netropsin (1 μg/ml) had no effect on the activity in vitro of both vegetative and sporulation holoenzymes on B. subtilis DNA; however, at this concentration the antibiotic inhibited the activity in vitro of the δ1 enzyme about 40%. These results indicate that the δ1 enzyme found in sporulation cells differs significantly from the holoenzymes found in vegetative and sporulating cells and from the vegetative δ enzyme.