ESCHERICHIA-COLI CAMP RECEPTOR PROTEIN-DNA COMPLEXES - 1 - ENERGETIC CONTRIBUTIONS OF HALF-SITES AND FLANKING SEQUENCES IN DNA RECOGNITION

In Escherichia coli, the cyclic AMP receptor protein (CRP) serves as a sensor of the intracellular level of cyclic AMP. At increasing concen trations of cyclic AMP, CRP becomes activated upon binding a cyclic AM P molecule. The activated CRP is capable of regulating the expression of more than 20 genes by binding to specific DNA sites. The specific D NA sequences recognized by CRP consist of two-half-sites of the consen sus sequence TGTGA......XCAXA. At present, the relative contributions of the two half-site and flanking sequences in the energetics of CRP r ecognition have not been quantitatively defined. A series of 20 DNA se quences was designed to dissect the contributions of individual half-s ites and flanking sequences using the natural gal P1 and lac P1 sequen ces 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 Inc P1 sequence, the combination of bo th half-sites leads to a > 100-fold increase in affinity compared to t hat of an individual half-site in CRP-DNA complex formation, The flank ing sequence of lac P1 exhibits a 10- and 0-fold enhancement in affini ty for CRP compared to that of a random sequence in the presence and a bsence of the two half-sites, respectively. The observations of the ga l P1 sequence differ from those of the lac P1 sequence. The combinatio n 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 contribut ion from the flanking sequence proximal to the conserved TGTGA motif. The total energetics of the gal-CRP complex formation is essentially d ue to the presence of the conserved half-site and its adjacent flankin g sequence. Thus, the relative contributions of the half-site and flan king sequences to the energetics of DNA recognition are operon specifi c.