Migration Retardation Assay and DNA Looping - Repression and the lac Operon

The Migration Retardation Assay and DNA Looping

Earlier it was mentioned that DNA can be subjected to electrophoresis under conditions compatible with protein binding. For DNA fragments in the size range of 50 to about 2,000 base pairs, the binding of a protein significantly retards the migration. Therefore, free DNA and protein-DNA complexes may easily be separated by electrophoresis and identi-fied by staining or autoradiography. An additional virtue of the migration retardation assay is the fact that protein and DNA can be incubated in buffers containing physiological concentrations of salt, on the order of 50 mM KCl. Then, the electrophoresis can be performed at very low salt concentration. As discussed earlier, salt has a dramatic effect on the affinity with which most proteins bind to DNA. At low salt concentrations, most proteins’ affinity is much higher than at physi-ological salt concentrations. This greatly reduces the dissociation rate of the proteins. Additionally, the presence of the gel surrounding the protein-DNA complex cages the protein and further reduces its effective dissociation rate during electrophoresis. These features make the gel migration retardation assay particularly useful for the study of protein-DNA interactions as electrophoresis “freezes” a particular solution condition.

Not surprisingly, the lac repressor-operator interaction can be stud-ied with the migration retardation assay. The tetrameric lac repressor can bind with two of its subunits to the main operator at the promoter. Repressor’s other two subunits are free to bind to either of the pseudo-operators that are located one hundred and four hundred base pairs to either side. Such a double binding by a single repressor tetramer forms

Figure 11.8 Migration retardation assay withlacrepressor and DNA contain-ing two operators. As repressor concentration is increased, loops, then linear structures are formed. At high DNA concentrations, sandwiches form.

a DNA loop. In vivo this looping reaction is facilitated by supercoiling. The same looping reaction can be facilitated in vitro by using a linear DNA fragment containing two strong repressor-binding sites separated by 100 base pairs. Incubation of such DNA with low concentrations of repressor permits binding of single tetramers to DNA molecules. These form DNA loops so that each tetramer contacts two operators from the same DNA molecule. Incubation at higher repressor concentrations forces a separate repressor tetramer onto each operator, and incubation at high DNA concentrations forms structures in which repressors join two DNA molecules in a sandwich structure (Fig. 11.8).