In Escherichia coli, the cyclic AMP receptor protein (CRP) serves as a sensor of the intracellular level of cyclic AMP. At increasing concentrations of cyclic AMP, CRP becomes activated upon binding a cyclic AMP molecule. The activated CRP is capable of regulating the expression of more than 20 genes by binding to specific DNA sites. The specific DNA sequences recognized by CRP consist of two-half-sites of the consensus sequence TGTGA......XCAXA. At present, the relative contributions of the two half-site and flanking sequences in the energetics of CRP recognition have not been quantitatively defined. A series of 20 DNA sequences was designed to dissect the contributions of individual half-sites and flanking sequences using the natural gal P1 and lac P1 sequences as the initial targets. The binding of CRP to these DNA sequences was monitored by fluorescence anisotropy. None of the individual half-sites or flanking sequences contributes more to the binding energetics than a random sequence. In the lac P1 sequence, the combination of both half-sites leads to a > 100-fold increase in affinity compared to that of an individual half-site in CRP-DNA complex formation. The flanking sequence of lac P1 exhibits a 10- and 0-fold enhancement in affinity for CRP compared to that of a random sequence in the presence and absence of the two half-sites, respectively. The observations of the gal P1 sequence differ from those of the lac P1 sequence. The combination of both half-sites exhibits no significant increase in affinity, but the flanking sequence exhibits a 100-fold enhancement in the presence of the two half-sites. However, there is a disproportionate contribution from the flanking sequence proximal to the conserved TGTGA motif. The total energetics of the gal-CRP complex formation is essentially due to the presence of the conserved half- site and its adjacent flanking sequence. Thus, the relative contributions of the half-site and flanking sequences to the energetics of DNA recognition are operon specific.
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