The characterization of internal promoters in the Bacillus subtilis riboflavin biosynthesis operon

The transcription start sites of two internal promoters, the P2 and P3 promoters, in the Bacillus subtilis riboflavin biosynthesis operon were identified by primer extension. Putative -35 and -10 sequences that are recognized by the vegetative delta(70) subunit of RNA polymerase have been found upstream of the P2 and P3 transcription start sites. The relative strengths of the P1, P2, and P3 promoters were determined by cloning these promoters into the pDG268 expression vector. It was shown that the transcriptional activity of the P3 promoter is approximately fivefold higher as compared with P1, the major promoter, whereas P2 promoter activity is lower by almost two orders of magnitude. Real-time PCR demonstrated that unlike the P1 promoter, P2- and P3-driven expression is not regulated by flavins.