ABSTRACT
How the sequence of a response element affects the binding of a transcription factor and, ultimately, the differential rate of transcription of genes under its control is not well-understood. In the case of the p73 transcription factor, it binds to >200 response elements to trigger developmental, cell arrest, and apoptotic pathways. The p73 response elements match the 20 bp consensus sequence of the p53 response elements that are formed by two 10 bp half-sites, where each half-site is an inverted repeat of two 5 bp quarter-sites. Using sedimentation velocity and fluorescence anisotropy experiments, we studied how systematic variations in the sequence of a half-site response element modify the DNA binding affinity of the p73 DNA-binding domain. We observed that each nucleotide position in the response element has a different influence in determining the binding of the p73 DNA-binding domain. The cytosine in the fourth position of each quarter-site is the largest determinant of DNA binding, followed by the nucleotide in the fifth position, and last, the first three positions show a slight regulatory preference for purines. Together with previous structural and functional results, our data suggest a hierarchical model of binding in which some nucleotide positions in the response element are more important than others in determining the binding of the transcription factor.