Supercoiling response of the lac ps promoter in vitro

Abstract

The rate of open promoter complex formation was measured on lac ps promoter DNA templates differing in negative superhelicity. The templates ranged from fully relaxed to those with numbers of superhelical turns exceeding that of form I plasmid DNA. The observed transcription response had two clearly distinguished phases: an initial rapid rise in rate followed eventually by a precipitous inhibition. The stimulation phase involved a nearly 40-fold increase in rate, which peaks at superhelical densities near that of isolated form I plasmid DNA. The introduction of more negative superhelical turns leads to inhibition. The magnitude of the response and the observation of both increases and decreases suggest that minor differences in superhelicity in vivo could lead to significant increases or decreases in transcription rate. The increase in rate was found to be directly proportional to the free energy of supercoiling; that is, to the square of the superhelical density. We suggest that the energy may be used both for enhanced DNA melting and for changes in DNA structure that alter the helical "face" with which RNA polymerase must interact. A quantitative method is presented that allows simple estimation of differences in the supercoiling response among promoters, both in the presence and in the absence of added factors.