We recently proposed a model for I kappa B alpha-mediated molecular stripping of NF kappa B from transcription sites. I kappa B alpha was shown experimentally to form a transient ternary complex with DNA-bound NF kappa B, but the mechanism by which the I kappa B alpha accelerates dissociation of the NF kappa B from the DNA was unknown. In this paper we construct and compute free energy profiles for the wild-type I kappa B alpha-mediated molecular stripping reaction of NF kappa B from DNA and compare with that for a mutant of I kappa B alpha bearing a charge-neutralized PEST. The differences in the free energy profile for stripping originate from the frustrated electrostatic interactions between the negatively charged PEST and the DNA. The PEST occupies two different conformations in the NF kappa B-I kappa B alpha binary complex, one of which occupies the DNA-binding cavity. Specific interactions with positively charged residues in the N-terminal domains of both p50 and p65 apparently draw the domains closer together hindering reassociation of DNA. Comparison with the charge-neutralized mutant reveals that all of these functional consequences result from the negative charges in the PEST sequence of I kappa B alpha.