Orientation of OmpR monomers within an OmpR : DNA complex determined by DNA affinity cleaving

Escizerichia coli OmpR is a transcription factor that regulates the differe ntial expression of the porin genes ompF and ompC. Phosphorylated OmpR bind s as a dimer to a 20-bp region of DNA consisting of two tandemly arranged 1 0-bp half-sites. Expression of the ompF gene is achieved by the hierarchica l occupation of three adjacent 20-bp binding sites, designated F1, F2, and F3 and a distally located site, F4. Despite genetic, biochemical, and struc tural studies, specific details of the interaction between phosphorylated O mpR and the DNA remain unknown. We have linked the DNA cleaving moiety o-ph enanthroline-copper to eight different sites within the DNA binding domain of OmpR in order to determine the orientation of the two OmpR monomers in t he OmpR:F1 complex. Five of the resulting conjugates exhibited DNA cleaving activity, and four of these yielded patterns that could be used to constru ct a model of the OmpR:F1 complex. We propose that OmpR binds asymmetricall y to the F1 site as a tandemly arranged dimer with each monomer having its recognition helix in the major groove. The N-terminal end of the recognitio n helix is promoter-proximal and flanked by "wings" W1 and W2 positioned pr oximally and distally, respectively, to the transcription start site of omp F. We further propose that the C-terminal end of the recognition helix make s the most extensive contacts with DNA and predict bases within the F1 site that are sufficiently close to be contacted by the recognition helix. (C) 1999 Academic Press.