Selective binding of Escherichia coli RNA polymerase to topoisomers of minicircles carrying the TAC16 and TAC17 promoters.

We have studied the effect of DNA supercoiling on open complex formation by Escherichia coli RNA polymerase at the TAC16 and TAC17 promoters. A two-dimensional gel retardation assay was used to measure the relative rate of association between RNA polymerase and the TAC promoters on individual topoisomers. Plasmid DNAs usually have several promoters that complicate the analysis of RNA polymerase binding to only one of them. We avoided this problem by using minicircles of DNA carrying only a single promoter. These were generated in vivo by the site-specific recombination system of bacteriophage lambda. Both the TAC16 and TAC17 promoters exhibited a biphasic response to negative supercoiling. Between superhelical densities of 0 and -0.04, increases in the negative linking difference favored binding. Beyond -0.04, either no effect on binding (TAC16) or a slight inhibition of binding (TAC17) was observed for increases in the negative linking difference. The unwinding at the rate-limiting step was calculated for both TAC promoters at moderate superhelical densities. This unwinding was found to be a fraction (about 20-30%) of the total unwinding measured by topoisomerase I relaxation (about one turn). In addition, the 1-base pair difference in spacer length between the TAC16 and TAC17 promoters resulted in different extents of activation and inhibition by negative supercoiling.