Influence of negative supercoiling and of the proximity of left-handed Z-DNA on the Escherichia coli lactose repressor-operator interaction.

The influence of negative supercoiling and of flanking (dC-dG) tracts in either right-handed B- or left-handed Z-structures on the interaction of the Escherichia coli lac repressor was investigated. The operator was embodied within the lac control sequence, which was 95, 59, or 29 base pairs in length. Thus, the (dC-dG) regions (in either B- or Z-conformations) were at different distances from the repressor-binding site. Surprisingly, the presence of the promoter sequence (-59 to -20 relative to the +1 transcription start site) of the lac operator region increases the binding affinity of lactose repressor to the operator at high negative supercoil densities. This influence of the promoter region on the binding was abolished when the flanking (dC-dG) tracts were in the left-handed Z-DNA conformation. In contrast, minimal differences in the binding affinities were observed between plasmids containing shorter operator fragments (59 or 29 base pairs), whether the flanking (dC-dG) tracts were in right-handed B- or left-handed Z-forms. The promoter region may be directly involved in the repressor-operator complex in a previously unrecognized manner or may exert structural influence on the operator region. In general, increasing the amount of negative supercoiling increases the binding affinity and decreases the dissociation rate constants for the three operator-containing fragments, both with and without flanking Z-DNA tracts. Thus, the lac operator region possesses a previously unrecognized structural pliability, as influenced by negative supercoiling and neighboring sequences and/or conformations, which modulates its biological properties.