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 S1 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.(ABSTRACT TRUNCATED AT 250 WORDS)