SEQUESTRATION STABILIZES LAC REPRESSOR-DNA COMPLEXES DURING GEL-ELECTROPHORESIS

The gel electrophoresis mobility shift assay is widely used for both q ualitative and quantitative characterization of protein-nucleic acid i nteractions. Often it is found that protein-nucleic acid complexes per sist within gels for much longer than would be expected on the basis o f their free solution lifetimes. Excluded volume and matrix-interactio n mechanisms have been proposed to account for the enhanced stabilitie s of complexes within gels. To test these mechanisms, we have investig ated the influences of gel composition and concentration on the pseudo first-order dissociation kinetics of complexes containing the Escheri chia coli lactose (lac) repressor protein and lactose promoter DNA. In both polyacrylamide and agarose gels, dissociation rates were slower than those in free solution and decreased with increasing gel concentr ation. This result is inconsistent with mechanisms of stabilization th at require specific interactions with the gel matrix. Under standard r eaction conditions, free solution values of k(diss) were proportional to [DNA](0.83+/-0.11), while in 10% polyacrylamide gels k(diss) values were proportional to [DNA](0.48+/-0.09). These results suggest that t he lifetimes of lac repressor-DNA complexes in free solution are limit ed by their encounter frequency with molecules of DNA or with protein- DNA complexes; some or all of the stabilization observed in gels may b e due to a reduction in this frequency. (C) 1997 Academic Press.