Measurement of the binding of transcription factor Sp1 to a single GC box recognition sequence

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Abstract
The equilibrium constant was determined for the binding of the transcription factor Sp1 to a single consensus GC box DNA recognition site, (5′-GGGGCGGGGC-3′). For these experiments, single copies of the recognition site were synthesized and cloned in a standard plasmid background. Binding was measured either by a footprinting assay modified so that the binding reaction was at equilibrium, or by a gel mobility shift assay. The concentration of active Sp1 in the reactions and the dissociation constant were determined by computer-assisted fitting to theoretical curves. Values for the dissociation constant obtained in different experiments ranged from 4.1 × 10−10 M to 5.3 × 10−10 M. Several variants of the consensus recognition site were also tested. An A-substituted variant (5′ -GGGGAAGGGGC-31) and a T-substituted variant (5′-GGGGTGGGGC-3′) were bound 3-fold and 6-fold more weakly than the consensus site, respectively. A G-substituted variant (5′-GGGGGGGGGC-3′) was bound at least 30-fold more weakly than the consensus site. These findings help distinguish between alternative models for Sp1-DNA recognition. They are consistent with the presence of specific hydrogen-bond contacts between Sp1 and the central C-G base pair, but provide no particular evidence to support a model where local DNA structure is the dominant factor in the interaction.