Minor groove recognition is important for the transcription factor PhoB: a surface plasmon resonance study

Abstract

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 (PhoB^DBD) was analyzed using surface plasmon resonance. In addition the two point mutants PhoB^DBDD196A and PhoB^DBDR219A were obtained and the DNA recognition in comparison to the wildtype PhoB^DBD was investigated. Aspartic acid 196 and arginine 219 mediate specific minor groove interactions. All results reveal that at high PhoB^DBD-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-PhoB^DBD. 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.