An operator at -280 base pairs that is required for repression of araBAD operon promoter: addition of DNA helical turns between the operator and promoter cyclically hinders repression

Abstract

A site has been found that is required for repression of the Escherichia coli araBAD operon. This site was detected by the in vivo properties of deletion mutants. In vitro protection studies with DNase I and dimethylsulfate showed that araC protein can specifically bind in this area to nucleotides lying at position -265 to -294 with respect to the araBAD operon promoter (PBAD) transcription start point. The previously known sites of protein binding in the ara operon lie between +20 and -160. Since the properties of deletion strains show that all the sites required for araBAD induction lie between +20 and -110, the new site at -280 exerts its repressive action over an unusually large distance along the DNA. Insertions of -16, -8, 0, 5, 11, 15, 24, and 31 base pairs of DNA between the new site and PBAD were constructed. Repression was impaired in those cases in which half-integral turns of the DNA helix were introduced, but repression was nearly normal for the insertions of 0, +11, and +31 base pairs.