The ability of curved DNA upstream of the -35 region to affect the interaction of Escherichia coli RNA polymerase and promoter DNA was examined through the use of hybrid promoters. These promoters were constructed by substituting the curved DNA from two Bacillus subtilis bacteriophage SP82 promoters for the comparable DNA of the bacteriophage λ promoters λp(R) and λp(L). The SP82 promoters possessed intrinsic DNA curvature upstream of their -35 regions, as characterized by runs of adenines in phase with the helical repeat. In vitro, the relative affinities of purified σ70-RNA polymerase for the promoters were determined in a competition binding assay. Hybrid promoters derived from λp(R) that contained curved DNA were bound by E. coli RNA polymerase more efficiently than was the original λp(R). Binding of E. coli RNA polymerase to these hybrid promoters was favored on superhelical DNA templates according to gel retardation analysis. Both the supercoiled and relaxed forms of the hybrid λp(L) series were better competitors for E. coli RNA polymerase binding than was the original λp(L). The results of DNase I footprinting analysis provided evidence for the wrapping of the upstream curved DNA of the hybrid λp(R) promoters around the E. coli RNA polymerase in a tight, nucleosomal-like fashion. The tight wrapping of the upstream DNA around the polymerase may facilitate the subsequent steps of DNA untwisting and strand separation.
ASJC Scopus subject areas
- Molecular Biology