The affinity and stoichiometry of DNA binding by Escherichia coli trp repressor were studied by electrophoresis in nondenaturing gels. The ability of trp repressor to retard the electrophoretic mobility of an operator DNA fragment depends on the pH of the gel system. Above the pI of the protein, little retardation of DNA is observed, although complex formation can be detected by other assays. As the pH of the gel is lowered, retardation is enhanced. The apparent dissociation constant for the interaction between trp repressor and trpEDCBA operator fragments is 0.5 nM under the conditions used here. Nonspecific binding occurs with only about 200-fold weaker affinity. The stoichiometries of specific and nonspecific complexes were determined directly by using trp repressor labeled in vivo. High-affinity operator binding requires a single dimer of trp repressor. DNase I-protection analysis ("footprinting") was used to confirm the dissociation constants and to locate the binding site.
|Original language||English (US)|
|Number of pages||5|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - Feb 1988|
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