Measurement of the binding of transcription factor Sp1 to a single GC box recognition sequence.
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'-GGGGAGGGGC-3') 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.
Letovsky
J
J
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309.
Dynan
W S
WS
D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D014644:Genetic Variation; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D009710:Nucleotide Mapping; D009841:Oligonucleotides / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
D009841:Oligonucleotides; D014157:Transcription Factors; D004247:DNA