Effect of polyadenine-containing curved DNA on promoter utilization in Bacillus subtilis

Abstract

The effect of DNA upstream of the -35 region on promoter function was examined using two promoters isolated from the Bacillus subtilis bacteriophage SP82. The affinity of RNA polymerase for the two promoters in vitro differed significantly. For each promoter the nucleotide sequence of the upstream DNA was characterized by the presence of successive runs of adenines with a 10-11-base pair periodicity. DNA fragments with the polyadenine-containing upstream DNA displayed aberrant electrophoretic mobilities when analyzed on polyacrylamide gels indicative of curved DNA. A series of mutant promoters in which the upstream DNA was deleted or altered was constructed. The curved DNA upstream of the -35 region was required for efficient RNA polymerase binding. Decreased in vitro transcription observed when the upstream DNA was deleted could be partially restored if the template was negatively supercoiled. Measurements of chloramphenicol acetyltransferase specific activity from B. subtilis strains carrying transcriptional fusions indicate that the curved upstream DNA stimulated transcription from the promoter with the weaker affinity for RNA polymerase. The curved DNA reduced the in vivo activity of the promoter with the strong affinity for RNA polymerase. One function of the curved upstream DNA may be to provide RNA polymerase-promoter interactions that facilitate open complex formation.