DNA Looping and Lac Repressor-CAP Interaction

The promoter regions of both eukaryotic and prokaryotic genes that are regulated contain multiple binding sites for transcription factors ( ). Interactions between and among regulatory proteins bound to promoter regions produce both positive and negative synergistic effects on gene expression in both eukaryotes and prokaryotes (1). The lac promoter of Escherichia coli has three binding sites for the lac repressor (lacR) centered at + 11(0), +400(02), and -82(03) relative to the transcription start site and one for the catabolite gene activator protein (CAP) centered at -61.5 (2). Lewis and his colleagues (3) proposed a detailed structural model based on co-crystal structures, with CAP bound inside a DNA loop stabilized by lacR bound to 01 and 03 on the opposite side of the DNA. We do not find this model to be correct. The lacR is a tetramer and contains two binding sites for lac operator DNA (4, 5). Our 2.6 A resolution crystal structure of the lacR core protein (6) showed two lacR dimers held together by an antiparallel fourhelix bundle of COOH-terminal co-helices, as suggested earlier by molecular genetic studies (7). Unanticipated, however, was the v-shaped, almost parallel arrangement of the two dimers that placed the DNA binding sites adjacent to each other on the same side of the tetramer (6). This arrangement of the binding sites has implications for the possible wrapping of multiple-operator-containing DNA that we explored (6) within the constraints of inter-operator spacing and footprinting data from MullerHill and his colleagues (5). Among the models considered (but not published because of the experiment described below) was the possibility that lacR (bound to 03) and CAP [bound to its site 20.5 base pairs (bp) away] would interact favorably side by side, as has been demonstrated to occur in the Deo promoter, where CAP interacts with CytR (a repressor homologous to lacR), which is bound two turns of DNA away (8). Previously published work by Hudson and Fried (9) demonstrates cooperativity between lacR and CAP when they bind the lac operator region. However, when Hudson and Fried performed DNA footprinting experiments on ternary IacRCAP-DNA complexes, they did not detect protection of 03, although 01 and CAPI were efficiently protected. To find out whether CAPI and O3 are bound cooperatively by their respective regulators, we performed gel retardation assays with a double-strand oligonucleotide spanning the corresponding region of the lac promoter (top sequence of Fig. IA). We detected no ternary complex between lacR, CAP, and DNA, although either protein bound DNA efficiently to form the respective binary complex (Fig. 2A). When either of the binary complexes was combined with variable concentrations of the third protein, we observed competition for the DNA probe, present in limiting concentrations. We obtained similar results when we repeated these experiments with oligonucleotides bearing sequences of various affinities for CAP and lacR, separated by 20.5 bases (10). We designed an oligonucleotide that carried symmetrized versions of both lacR and CAP-binding sequences, identical to those used to solve the structure of the DNA complexes of these proteins (3, 11), except for a five-base "overlap" (bottom