Repression oflacPromoter as a Function of Distance, Phase and Quality of an AuxiliarylacOperator

Abstract The tetrameric Lac repressor can bind simultaneously to two lac operators on the same DNA molecule, thereby including the formation of a DNA loop. We investigated the phasing dependence of DNA loop formation between lac operator O 1 and an auxiliary ideal lac operator (O id ) on the bacterial chromosome, with inter-operator distances varying from 57.5 to 1493.5 bp. Repression of a CAP-independent lac UV5 promoter by O 1 at its natural position increased up to 50-fold in the presence of an optimally positioned auxiliary O id . Repression values alternated between local maxima and minima with a periodicity of 11.0 to 11.3 bp, suggesting that the chromosomal helical repeat is in this range in vivo . Repression increased significantly with decreasing inter-operator DNA length, indicating that the local Lac repressor concentration at O 1 is crucial for tight repression. Maximal repression, attributed to stable DNA loop formation, was obtained at an operator spacing of 70.5 bp. Other repression maxima occurred at operator distances of 92.5 and 115.5 bp, corresponding to natural operator spacings in the lac and in the gal operon, respectively. Substitution of the auxiliary O id with the weaker binding lac operator O 3 lowered repression efficiency, presumably due to the reduced local concentration of Lac repressor.