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 (E sigma 29) that contains a 29,000 Mr 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 E sigma 29 has been limited by the small number of cloned promoters that are recognized by E sigma 29. We have determined the nucleotide sequence of a recently isolated promoter (G4) that is used exclusively by E sigma 29 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 E sigma 29 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 E sigma 29 in in-vitro competition experiments. The effects of these mutations in G4 on its use by E sigma 29 support a model in which E sigma 29 utilizes its cognate promoters by interacting with unique nucleotide sequences at the -10 region and near the -35 region of these promoters.