Antibody directed against Bacillus subtilis rho factor purified by sodium dodecyl sulfate slab gel electrophoresis. Effect on transcription by RNA polymerase in crude extracts of vegetative and sporulating cells

Abstract

Antibody directed against rho factor from vegetative Bacillus subtilis was prepared by immunizing a rabbit with denaturated rho polypeptide isolated by electrophoresis of partially purified DNA-dependent RNA polymerase on a sodium dodecyl sulfate-polyacrylamide slab gel. Antiserum to rho reacted specifically with native rho polypeptide but not with core RNA polymerase as judged by complement fixation and by an immunodiffusion assay. Anti-rho antibody also inhibited the ability of rho to stimulate transcription of phage phie DNA but failed to inhibit transcription of poly(dA-dT) by core enzyme. Specific antibody was also raised against a mixture of the beta and beta' subunits of RNA polymerase purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The effect of the anti-rho gamma-globulin on the transcription of phage phie DNA by RNA polymerase in crude extracts of vegetative and sporulating cells was examined. Anti-rho antibody markedly inhibited the transcription of phage DNA by RNA polymerase partially purified from vegetative bacteria by ammonium sulfate fractionation but had little effect on transcription of the phage DNA template by enzyme from sporulating cells. Addition of purified rho to a vegetative extract that had been depleted of rho by treatment with the anti-rho antibody restored active transcription of phage DNA. However, addition of purified rho to an antibody-treated extract of sporulating cells had little effect on phie RNA synthesis. These findings suggest that sporulating cells contain a component that interferes with the activity of the rho subunit of RNA polymerase.