Rotational orientation of upstream curved DNA affects promoter function in Bacillus subtilis

Abstract

The Alu156 promoter isolated from the Bacillus subtilis bacteriophage SP82 is dependent on curved DNA upstream of the -35 region for efficient function. Short DNA insertions of 6-29 base pairs were used to simultaneously change the linear placement and rotational orientation of this curved DNA relative to the -35 region. When these mutant promoters were analyzed in vivo using transcriptional fusions with a chloramphenicol acetyltransferase gene, changes in the rotational orientation of the curved DNA correlated with changes in promoter function. The most efficient mutant promoters contained insertions of 11 and 21 base pairs, and insertions of 15 and 25 base pairs resulted in the least efficient mutant promoters. The importance of the proper rotational alignment of the curved DNA to promoter activity was also observed in vitro at the level of transcription of RNA polymerase binding. Based on the electrophoretic mobilities of DNA fragments containing the various insertion mutant promoters, there was a second region of curved DNA downstream of the insertion point. The findings are consistent with the idea that the curved DNA deflects the helix back toward the promoter-bound RNA polymerase molecule to allow the enzyme to interact directly with upstream DNA. These interactions are proposed to structure the DNA for the formation of the open promoter complex.