Interaction of Bacillus subtilis RNA polymerase core with two specificity-determining subunits. Competition between...

Gene activity in the development of phage SPO1 is transcriptionally regulated. Early viral genes are transcribed by the major vegetative cell RNA polymerase (E. sigma). Middle viral genes are transcribed by RNA polymerase core (E) bearing the SPO1 gene 28 protein (gp28) instead of sigma. This paper deals with the competitive interactions of sigma and gp28 with E which must, at least in part, be involved in the ability of viral middle gene expression to succeed early gene expression. An in vitro assay has been developed for determining the proportions of early (E. sigma) and SPO1 middle (E.gp28) Bacillus subtilis RNA polymerase in mixtures. The assay, which is based on the transcription of two well studied SPO1 restriction fragments followed by gel electrophoresis and quantitation of the E. sigma and E.gp28 transcription products, has been used to study the interactions of sigma and gp28 with RNA polymerase core. These subunits are found to be capable of competitively excluding each other. The binding competition is sensitive to ionic strength, with sigma being more effective below 0.2 total ionic strength and gp28 being more effective at higher values. The outcome of competition is also dependent on the order of addition of subunits and the reconstitution kinetics have been studied. The subunit competition is rather insensitive, particularly at higher ionic strength, to temperature. Gp28 can convert E. sigma to E.gp28 but the reverse reaction occurs much less efficiently. The B. subtilis delta protein biases the sigma-gp28 competition in favor of the sigma subunit. the implications of these results for the physiological transition of transcriptional specificity during SPO1 development are discussed.