Purification and characterization of RNA polymerase holoenzyme (E sigma B) from vegetative-phase mycelia of Streptomyces griseus

Abstract

RNA polymerase was purified from vegetative-phase mycelia of Streptomyces griseus by a series of ion-exchange chromatographies. By western blot analysis using antiserum against S. coelicolor HrdB, which is a principal sigma factor (sigma(hrdB)), the purified holoenzyme was found to contain sigmaB (=sigma(hrdB)) of S. griseus. Significant amounts of HrdB protein were, however, eluted from the DEAE column at lower concentrations of KCl than that required for for elution of the holoenzyme containing sigmaB, suggesting that sigmaB is dissociated from the core enzyme, or an excess amount of sigmaB exists in S.griseus cells. The holoenzyme containing sigmaB (EsigmaB) transcribed in vitro the dagA promoter of S. coelicolor, and the hardB and hsp70 promoters of S. griseus, suggesting that it is involved in transcription of the essential genes. EsigmaB may be a major form of RNA polymerase holoenzyme in the growing phase of S. griseus.