In vitro repression of the gal promoters by GalR and HU depends on the proper helical phasing of the two operators.

Repression of transcription initiation from the two gal promoters, P1 and P2, requires binding of GalR protein to two flanking operators, O(E) and O(I), binding of HU to a site, hbs, located between the two operators, and supercoiled DNA template. Previous experiments suggested that repression involves the interaction of two DNA-bound GalR proteins, which generates a 113-bp DNA loop encompassing the promoter region. Interaction between two DNA-bound proteins would be allowed if the binding sites on DNA are properly aligned. To test the idea that the observed repression of gal transcription in vitro is mediated by DNA looping, we investigated the effect of changing the relative angular orientation of O(E) and O(I) in the DNA helix. We found that repression is a periodic function of the distance between the two operator sites. Since repression recurred commensurate with DNA helical repeat, we conclude that the observed in vitro repression is mediated by DNA looping and the in vitro conditions reflect the in vivo situation.