Spectroscopic studies on lambda cro protein-DNA interactions.

Abstract

Spectroscopic (circular dichroism and fluorescence) and thermodynamic studies were conducted on lambda Cro-DNA interactions. Some base substitutions were introduced to the operator and the effects on the conformation of the complex and thermodynamic parameters for dissociation of the complex were examined. It was found that, (1) in the specific binding of Cro with DNA which has a (pseudo) consensus sequence, DNA is overwound, while in non-specific binding it is unchanged, or rather unwound; (2) substitution of central base-pairs or the introduction of a mismatched base-pair at the center of the operator reduces the extent of DNA conformational change on Cro binding and lessens the stability of the Cro-DNA complex, even though there is apparently no direct interaction between Cro and DNA at these positions; (3) stability of the complex increases with the degree of DNA conformational change of the same type during binding; (4) in some cases of specific binding, there are three states in the dissociation of the complex as observed by salt titration: two conformational states for the complex depending on salt concentration and, in non-specific binding, dissociation is a two-state transition; (5) the number of ions involved in interactions between Cro and 17 base-pair DNA is about 7.7 for NaCl titrations; (6) dissociation free energy prediction of the Cro-DNA complex by simple addition of the dissociation free energy change of a single base-pair substitution agrees with our experimental results when DNA overwinding occurs during binding, i.e. in specific binding.