ASSEMBLY OF THE ARC REPRESSOR OPERATOR COMPLEX - COOPERATIVE INTERACTIONS BETWEEN DNA-BOUND DIMERS

Arc repressor, a member of the beta-ribbon family of DNA binding proteins, binds to its 21-base-pair operator as a tetramer. Here, the Arc dimer is shown to bind specifically to DNA fragments containing operator half-sites, and the equilibrium and kinetic constants for these reactions are determined. DNA-bound dimers are also shown to be transient intermediates in association experiments, indicating that assembly of the Arc tetramer-operator complex occurs by sequential addition of dimers to operator half-sites. When the left or right operator half-site is occupied by an Arc dimer, cooperative interactions increase the affinity of the second dimer by approximately 5900-fold [DELTADELTAG = -5.1 (+/-0.5) kcal/mol]. This increase in affinity is largely caused by an increase in the half-life of the complex; ''non-cooperatively'' bound dimers dissociate with a half-life of a few seconds while ''cooperatively'' bound dimers have half-lives of more than 1 h.