Dimeric lac repressors exhibit phase-dependent co-operativity

Transcription of the lac operon in Escherichia coli is repressed by the binding of Lac repressor (LacR) to lac operator O-1, a pseudo-palindromic sequence centred 11 bp downstream of the transcription start. Full repression of the wild-type promoter by wild-type, tetrameric LacR requires the presence of at least two operator sequences that must not only be in close proximity to O-1, 401 bp and 92 bp for the auxiliary operators O-2, and O-3, respectively, but must also be present on the same side of the DNA helix. LacR mutants lacking the C-terminal heptad repeat and thus only capable of dimer formation still repress, but at a much reduced level. Their repression of the inc promoter is comparable to repression by tetrameric LacR when both auxiliary operators are destroyed. We have examined the residual repression, by dimeric LacR, of a series of constructs containing a CAP-independent promoter and two lac operators, O-1, and O-id, separated by a series of spacers increasing in size by single base-pair increments. Surprisingly, repression of these constructs still exhibits phase dependence. The periodicity of maxima is similar to the helical repeat of DNA in vivo, as measured by phase-dependent repression with tetrameric LacR, although the magnitude of repression is much smaller than that obtained in previous experiments with tetrameric LacR. Two additional variants of dimeric LacR with altered C termini that were tested also show phase dependence. Control experiments show that the presence of O-1, is required for repression in this system. In the absence of O-1, occupancy of the auxiliary operator does not lead to repression. The magnitudes of repression maxima correlate best with the overall basic nature of the C terminus. Weak, unspecific contacts by this region with DNA seem sufficient to explain the observed periodicity. It remains to be seen wether additional factors are also involved in this residual repression. (C) 1998 Academic Press.