Nucleotide sequences that define promoters that are used by Bacillus subtilis sigma-29 RNA polymerase

There are at least five different forms of RNA polymerase holoenzyme in Bacillus subtilis. These enzymes differ in their sigma subunit and their specificity for promoter utilization. One form of RNA polymerase (Eσ²⁹) that contains a 29,000 M_r sigma appears in B. subtilis about two hours after the initiation of endospore formation. The determination of the nucleotide sequences that govern utilization of promoters by Eσ²⁹ has been limited by the small number of cloned promoters that are recognized by Eσ²⁹. We have determined the nucleotide sequence of a recently isolated promoter (G4) that is used exclusively by Eσ²⁹ both in vitro and in vivo. The start-point of transcription was identified by S₁ nuclease mapping and dinucleotide priming experiments and the probable promoter element was sequenced. We compared the sequence with that of six promoters that are used to varying degrees in vitro by Eσ²⁹ and found these sequences to be highly conserved at the -10 and near the -35 regions of these promoters. Single base substitutions were generated at positions -12, -15 and -36 of the G4 promoter and assayed for their influence on utilization by Eσ²⁹ in in-vitro competition experiments. The effects of these mutations in G4 on its use by Eσ²⁹ support a model in which Eσ²⁹ utilizes its cognate promoters by interacting with unique nucleotide sequences at the -10 region and near the -35 region of these promoters.