Coupled Energetics of λ cro Repressor Self-Assembly and Site-Specific DNA Operator Binding I: Analysis of cro Dimerization from Nanomolar to Micromolar Concentrations

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Abstract
The cro repressor from bacteriophage λ is an important and classical transcription regulatory protein that binds DNA operator sites as a dimer. Therefore, a complete understanding of gene regulation by cro requires knowledge of the coupled energetics of its protein dimerization and site-specific DNA binding. A method is described by which cro repressor can be labeled in vivo with [35S]methionine to a specific activity of 2 × 1015 cpm/mol. As a prelude to binding studies, the association equilibrium of cro was determined over the range 10-9−10-3 M using large-zone analytical gel chromatography with radiolabeled repressor. The data are best described by a monomer−dimer stoichiometry with an equilibrium constant of 3.07 (±1.08) × 106 M-1 total cro monomer. Stokes radii for monomers and dimers were evaluated from the resolved gel partition coefficients. Under the conditions employed in this study (10 mM Bis-Tris, 200 mM KCl, 2.5 mM MgCl2, 1 mM CaCl2, 100 μg/mL BSA, pH 7.0, 20 °C), self-association of cro to species with assembly states greater than dimers is not observed.