Sequence Correlations between Cro Recognition Helices and Cognate OR Consensus Half-sites Suggest Conserved Rules of Protein–DNA Recognition

The OR regions from several lambdoid bacteriophages contain the three regulatory sites OR1, OR2 and OR3, to which the Cro and CI proteins can bind. These sites show imperfect dyad symmetry, have similar sequences, and generally lie on the same face of the DNA double helix. We have developed a computational method, which analyzes the OR regions of additional phages and predicts the location of these three sites. After tuning the method to predict known OR sites accurately, we used it to predict unknown sites, and ultimately compiled a database of 32 known and predicted OR binding site sets. We then identified sequences of the recognition helices (RH) for the cognate Cro proteins through manual inspection of multiple sequence alignments. Comparison of Cro RH and consensus OR half-site sequences revealed strong one-to-one correlations between two amino acids at each of three RH positions and two bases at each of three half-site positions (H1→2, H3→5 and H6→6). In each of these three cases, one of the two amino acid/base-pairings corresponds to a contact observed in the crystal structure of a lambda Cro/consensus operator complex. The alternate amino acid/base combinations were rationalized using structural models. We suggest that the pairs of amino acid residues act as binary switches that efficiently modulate specificity for different consensus half-site variants during evolution. The observation of structurally reasonable amino acid-to-base correlations suggests that Cro proteins share some common rules of recognition despite their functional and structural diversity.