From footprint to toeprint: A close-up of the DnaA box, the binding site for the bacterial initiator protein DnaA

The Escherichia coli DnaA protein binds as a monomer to the DnaA box, a 9 bp consensus DNA sequence: 5'-TTA/(T)TNCACA. To assess the contribution of individual bases to protein binding we probed the DnaA-DnaA box complex with the uracil-DNA glycosylase (UDG) footprinting technique, (i) dU at the positions of T-2, T-4, T-7' Or Ts completely inhibits DnaA binding to the DnaA box. At these positions the methyl groups of the thymine residues are essential for successful DnaA binding, indicating protein contact with the major groove. Additionally they are positioned exactly on one side of the helix. (ii) dU at the position of T-1 or at three T residues adjacent to the 9 bp core sequence of the DnaA box allows DnaA binding. These positions are protected from UDG digestion as revealed by the footprint assay. (iii) dU at the position of T-3' On the complementary strand of the box 5'-TTATCCACA was not protected from UDG digestion in DNA-DnaA complexes. Therefore, DnaA cannot contact the major groove at this position, In addition, a slight bend of the DnaA box towards UDG would help the enzyme to access this site.