MECHANISM OF INITIATION OF TRANSCRIPTION BY BACILLUS-SUBTILIS RNA-POLYMERASE AT SEVERAL PROMOTERS

The behavior of the major vegetative cell RNA polymerase of Bacillus subtilis, EσA, during initiation of transcription was compared to that of its Escherichia coli counterpart, Eσ70, at several promoters known to be actively transcribed by both RNA polymerases. Challenge experiments using heparin, restriction endonucleases, and competing promoter DNA under various conditions showed that, at several promoters, complexes with B. subtilis RNA polymerase formed in the absence of nucleoside triphosphates were unstable. These complexes produced DNase I footprints that were less extended than those produced by the E. coli enzyme at the same promoters. Further, in the presence of certain combinations of nucleoside triphosphates, conditions that allow production of abortive oligonucleotides, these B. subtilis RNA polymerase complexes remained dissociable. Thus, at these promoters, the B. subtilis enzyme interacted with the DNA and reached a catalytically active initial transcribing complex without becoming committed to the template. At these same promoters, E. coli RNA polymerase formed stable open complexes before forming any phosphodiester bonds. B. subtilis initial transcribing complexes also remained sensitive to the drug rifampicin until a later stage in the initiation process than did the corresponding E. coli complexes. At one promoter, B. subtilis EσA and E. coli Eσ70 behaved similarly, forming stable open complexes in the absence of any nucleoside triphosphates. © 1992.