Regulation of the initiation of gene transcription from the gal operon of Escherichia coli is activated by the binding of CAP (catabolite activator protein) to a site centered at base pair -41.5 relative to the SI start site of transcription. This operon is repressed by the specific binding of Gal repressor (GalR) to two operators, OE and OI, centered at -60.5 and +53.5, respectively. It has been proposed that this negative regulation results from the interaction of GalR dimers bound to OE and OI to form a protein-mediated“looped complex”[cf. Adhya, S. (1989) Annu. Rev. Genet. 23, 207-230], In order to test whether DNA-bound CAP would facilitate or inhibit the binding of GalR, the simultaneous binding of these proteins was studied by quantitative DNase I footprint titration analysis. These studies demonstrate that GalR binding is noncooperative in the presence and in the absence of CAP and that GalR and CAP bind to the gal operon independently. No evidence was found that CAP stabilizes a putative Gal repressor-mediated protein-DNA looped complex. It has been shown that the gal operon can be negatively regulated by the binding of Lac repressor (LacI) to a gal operon in which OE and OI were both modified to be recognized by LacI [Haber, R., & Adhya, S. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 9683-9687], In contrast to GalR, LacI binds to the chimeric gal operon with moderate cooperativity via the formation of a stable protein-DNA looped complex. In order to assess the effect of CAP on a Lacl-mediated looped complex at the gal operon, the simultaneous binding of these two proteins to DNA was studied. The simultaneous binding of CAP and Lac repressor has no significant effect on the cyclization probability of the Lacl-mediated“looped complex”. However, while no interaction was observed between bound CAP and LacI bound to the downstream operator, OI, an antagonistic interaction was observed when LacI was bound to the OE operator, which is adjacent to the CAP binding site. The observation that LacI binding at OEmodulates the DNase I hypersensitivity induced by CAP within its binding site suggests that the CAP-DNA interaction is disrupted, potentially decreasing the magnitude of the CAP-induced DNA bend. This disruption may account for the absence of an effect on the cyclization probability of the Lacl-mediated looped complex by CAP.
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