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 identified 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 physiological 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 studied 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 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. 1).

Fig1. Migration retardation assay with lac repressor and DNA containing two operators. As repressor concentration is increased, loops, then linear structures are formed. At high DNA concentrations, sandwiches form.

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مواضيع ذات صلة

Targeting of Mitochondrial Proteins

Intra-Golgi Transport and Protein Processing

Cotranslational Protein Translocation Into the Endoplasmic Reticulum

The Isolation and Structure of Operator

Repressor Binds to DNA: The Operator is DNA

Detection and Purification of lac Repressor

The Difficulty of Detecting Wild-Type lac Repressor

An Assay for lac Repressor

Proving lac Repressor is a Protein

The Role of Inducer Analogs in the Study of the lac Operon

Background of the lac Operon

Mutagenesis with Chemically Synthesized DNA