THEORETICAL-STUDIES ON THE MOBILITY-SHIFT ASSAY OF PROTEIN-DNA COMPLEXES

The theory of mass transport coupled to reversible macromolecular inte ractions under chemical kinetic control forms the basis for computer s imulation of the electrophoretic mobility-shift behavior of protein-DN A complexes. Model systems include (i) specific binding of a univalent protein molecule to a single site on the DNA molecule; (ii) the putat ive cage effect; (iii) cooperative binding to multiple sites; (iv) for mation of looped complexes of 1:1 and 2:1 stoichiometry; (v) noncooper ative and cooperative, nonspecific binding modes; and (vi) binding of dimerizing transcriptional factors to response elements of target gene s. Favorable comparison of simulated with experimental mobility-shift behavior indicates that the phenomenological mechanisms, whereby obser ved mobility-shift patterns are generated during electrophoresis, are embodied in the theory. These studies have provided guidelines for def initive interpretation of mobility-shift assays and for the design of experiments to develop a detailed unterstanding of the particular syst em under investigation.