RECOGNITION OF DNA-STRUCTURE BY 434 REPRESSOR

In complexes of bacteriophage 434 binding sites with 434 repressor the central 4 bp of the 14 bp site are not contacted by the protein, alth ough changes in these bases alter binding site affinity for the repres sor, Our previous data suggested that the ability of the noncontacted central bases to be overtwisted in repressor-DNA complexes governs aff inity of the binding site for 434 repressor, This idea was tested by e xamining the affinity of two central sequence variant 434 binding site s for 434 repressor as a function of binding site average twist, The 4 34 repressor preferred the relatively overwound binding site to the tw o more underwound forms, The greatest affinity enhancement resulting f rom increasing twist was observed with a binding site that is relative ly underwound and more resistant to twisting deformation. Consistent w ith the idea that 434 repressor overtwists its binding site upon DNA b inding, we show that 434 repressor is capable of binding to sites bear ing a single base insertion in their center (a 15mer), but binds poorl y to binding sites bearing central base deletions (12mer and 13mer), T he N-terminal dimer interface plays a large role in determining 434 re pressor central base preferences, Mutations in this interface eliminat e central base discrimination and/or site size preferences, These muta tions also lead to changes in the size of the repressor footprint on t he various sized DNA sites that are consistent with their binding char acteristics.