A dimer of AraC protein contacts three adjacent major groove regions of the araI DNA site.

Contact sites of AraC protein to the regulatory site araI of the Escherichia coli araBAD operon have been determined by the chemical-interference technique. DNA fragments were chemically modified an average of once per molecule, and fragments that no longer bound AraC were separated by gel electrophoresis from the DNA fragments still able to bind the protein. The contact sites were then determined by comparing the positions of modifications in the two DNA samples. Strong contacts were found with guanines in three consecutive major groove regions and the adjacent phosphates along one side of the DNA. The conserved bases of the AraC-binding DNA consensus sequence are also found in the same positions. The gel electrophoresis assay was used to determine the stoichiometry of binding, and AraC protein was found to bind the araI and araO1 regulatory sites as a dimer. Therefore, AraC appears to bind DNA differently from the other well-characterized regulatory proteins such as phage lambda repressor.