Supercoiling response of E. coli promoters with different spacer lengths.

The effect of negative supercoiling on a series of synthetic Escherichia coli promoters has been investigated. These promoters carry perfect consensus sequences at the -35 and -10 regions, but with different spacer lengths (Aoyama, T. et al. (1983) Nucleic Acids Res. 11, 5855-5864). Topoisomeric plasmids carrying these synthetic promoters were constructed, and their activities were compared by detecting in vitro transcripts with the probe-hybridization method. In the relaxed state, the one with 17 basepairs (bp) spacing showed the highest activity, and the activity steeply decreased both sides of the optimal spacing. Similar results have been observed by run-off transcription. By introducing negative superhelicity, the 17 bp spacing promoter showed a relatively little response to supercoiling. In contrast, the activities of those with 16 and 18 bp spacings were markedly stimulated by supercoiling, with the mean, negative superhelical density (-sigma) which gave the maximum activity being about the same for the 16-18 bp spacing promoters (-sigma = 0.03 to 0.04). The promoter with 19 bp spacing, which showed no activity in the relaxed state, exhibited a significant activity at higher superhelicities (-sigma = 0.06). Even the 20 bp spacing promoter showed some activity by increasing superhelicity, while the 15 bp spacing promoter did not. On the basis of these observations, possible mechanisms by which negative supercoiling of DNA stimulates the protomer activity are discussed.