The two-component regulatory system PhoR/PhoB induces the expression of several genes in response to phosphate starvation in Escherichia coli. In order to quantify these protein-DNA interactions and to study the time-resolved dynamics of the binding mechanism, the specific recognition of different oligonucleotide duplexes by the DNA-binding domain of PhoB (PhoBDBD) was analyzed using surface plasmon resonance. In addition the two point mutants PhoBDBDD196A and PhoBDBDR219A were obtained and the DNA recognition in comparison to the wildtype PhoBDBD was investigated. Aspartic acid 196 and arginine 219 mediate specific minor groove interactions. All results reveal that at high PhoBDBD-concentrations all recognition sequences of the pho box are occupied. Decreasing the protein amount results in a mixture of free oligonucleotides and DNA molecules occupied by two WT-PhoBDBD. Moreover, the SPR results indicate that both binding site segments, the TGTCA-motif and the A/T-rich minor groove, are essential for the binding process. A comparison of different regulons additionally proved the dependency of the recognition process on the base composition of the minor groove.
M. Ritzefeld, K. Wollschläger , G. Niemann , D. Anselmetti and N. Sewald, Mol. BioSyst., 2011, 7, 3132–3142.