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J. Biol. Chem., Vol. 266, Issue 21, 13661-13671, 07, 1991
DF Senear and M Brenowitz
We have investigated the question of whether the gel mobility-shift assay
can provide data that are useful to the demonstration of cooperativity in
the site-specific binding of proteins to DNA. Three common patterns of
protein-DNA interaction were considered: (i) the cooperative binding of a
protein to two sites (illustrated by the Escherichia coli Gal repressor);
(ii) the cooperative binding of a bidentate protein to two sites
(illustrated by the E. coli Lac repressor); and (iii) the cooperative
binding of a protein to three sites (illustrated by the lambda cI
repressor). A simple, rigorous, and easily extendable statistical
mechanical approach to the derivation of the binding equations for the
different patterns is presented. Both simulated and experimental data for
each case are analyzed. The mobility-shift assay provides estimates of the
macroscopic binding constants for each step of ligation based on its
separation of liganded species by the number of ligands bound. Resolution
of the binding constants depends on the precision with which the
equilibrium distribution of liganded species is determined over the entire
range of titration of each of the sites. However, the evaluation of
cooperativity from the macroscopic binding constants is meaningful only for
data that are also accurate. Some criteria that are useful in evaluating
accuracy are introduced and illustrated. Resolution of cooperative effects
is robust only for the simplest case, in which there are two identical
protein binding sites. In this case, cooperative effects of up to
1,000-fold are precisely determined. For heterogeneous sites, cooperative
effects of greater than 1,000-fold are resolvable, but weak cooperativity
is masked by the heterogeneity. For three-site systems, only averaged
pair-wise cooperative effects are resolvable.
Determination of binding constants for cooperative site-specific protein-DNA interactions using the gel mobility-shift assay
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
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