DNA supercoiling promotes formation of a bent repression loop in lac DNA

Titration experiments on supercoiled lac DNA show that one repressor tetramer can bind simultaneously to the primary lac operator and to the very weak lac pseudo-operator, located 93 base-pairs apart. The formation of this complex is accompanied by the appearance of an extreme hypersensitive site in a five base-pair sequence located approximately midway between the operators. This remote sequence is hypersensitive to attack by two different chemical probes, dimethyl sulfate and potassium permanganate, the latter of which is a new probe for distorted DNA. We interpret these results in terms of a complex in which lac repressor holds two remote operators together in a DNA loop. The formation of this bent DNA loop requires negative DNA supercoiling. In vivo, both lac operators bind repressor even though the presence of multiple operator copies has forced the two operators to compete for a limited amount of repressor. This suggests that the operator and pseudo-operator have similar affinities for repressor in vivo. Such similar affinities were observed in vitro only when DNA supercoiling forced formation of a repression loop.