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The transcription factor human GA-binding protein (hGABP) is composed of two subunits, the Ets-related hGABP␣, which binds to a specific DNA sequence, and either one of two hGABP␣-associated subunits, hGABP␤ or hGABP␥. The DNA-binding protein hGABP␣ cannot affect transcription by itself, but can modify hGABP-dependent transcription in vitro and in vivo in the presence of its associated subunits. In this study, co-transfection assays showed that the ratio of hGABP␤ to hGABP␥ affected transcription from a promoter containing hGABP binding sites. Biochemical analysis showed that they bind to hGABP␣ competitively, indicating that the ratio of hGABP␤ to hGABP␥ is important for hGABP complex formation. Kinetic analysis of the protein-protein interaction using the surface plasmon resonance system showed that hGABP␣ binds to hGABP␤ or hGABP␥ with similar equilibrium constants. Kinetic analysis of the DNA-hGABP interaction showed that the binding of hGABP␥ to hGABP␣ stabilized the interaction of hGABP␣ with its DNA binding site. In addition, the kinetic analysis revealed that this was due to a slower dissociation of the protein complex from the DNA. These results suggest that hGABP␣-associated subunits influence the DNA binding stability of hGABP␣ and regulate hGABP-mediated transcription by competing with each other.

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