Reengineering Cro protein functional specificity with an evolutionary code.

Cro proteins from different lambdoid bacteriophages are extremely variable in their target consensus DNA sequences and constitute an excellent model for evolution of transcription factor specificity. We experimentally tested a bioinformatically derived evolutionary code relating switches between pairs of amino acids at three recognition helix sites in Cro proteins to switches between pairs of nucleotide bases in the cognate consensus DNA half-sites. We generated all eight possible code variants of bacteriophage λ Cro and used electrophoretic mobility shift assays to compare binding of each variant to its own putative cognate site and to the wild-type cognate site; we also tested the wild-type protein against all eight DNA sites.