Purification, characterization, and transcriptional analyses of RNA polymerases from Rhodobacter sphaeroides cells grown chemoheterotrophically and photoheterotrophically

Abstract

RNA polymerase was purified from Rhodobacter sphaeroides cells grown both chemoheterotrophically and photoheterotrophically. Both preparations of polymerase were comprised of five major subunits designated: beta' (160,000 Da), beta (150,000 Da), sigma (93,000 Da), alpha (41,000 Da), and a 35,000-Da protein, designated epsilon. All five subunits of the polymerase isolated from photoheterotrophically grown cells were found to be serologically related to the major subunits (beta', beta, sigma, and alpha) of the RNA polymerase from Escherichia coli; however, only four of the subunits of the RNA polymerase isolated from chemoheterotrophically grown cells were serologically related to the four major E. coli RNA polymerase subunits. The enzyme isolated from photoheterotrophically grown cells had a lower specific activity and was considerably less stable than the enzyme isolated from chemoheterotrophically grown cells. However, RNA synthesis by both enzyme preparations was dependent upon the presence of DNA template and MgCl2, and the RNA synthetic activity was inhibited by rifampicin. The transcriptional activities of both samples of polymerase were studied using different DNA templates, and the sizes of the run-off products compared to expected values obtained from analysis of mRNA's produced in vivo. These results are discussed in light of DNA sequences expected to contain promoter-like regions as determined from in vivo studies.