Nucleic Acids Research, 1989, Vol. 17, No. 7 2639-2653
© 1989
MOLECULAR BIOLOGY |
Measurement of the binding of transcription factor Sp1 to a single GC box recognition sequence
Department of Chemistry and Biochemistry, Campus Box 215, University of Colorado Boulder, CO 80309, USA
*To whom correspondence should be addressed
Received December 14, 1988. Revised March 1, 1989. Accepted March 1, 1989.
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 x 10–10 M to 5.3 x 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.
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