Coupled energetics of λ cro repressor self-assembly and site-specific DNA operator binding I:: Analysis of cro dimerization from nanomolar to micromolar concentrations

The cro repressor from bacteriophage lambda 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 [S-35]methionine to a specific activity of 2 x 10(15) 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) x 10(6) 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 KCI, 2.5 mM MgCl2, 1 mM CaCl2, 100 mu g/mL BSA, pH 7.0, 20 degrees C), self-association of cro to species with assembly states greater than dimers is not observed.