The unphosphorylated receiver domain of PhoB silences the activity of its output domain.

PhoB is the response regulator of the Pho regulon. It is composed of two distinct domains, an N-terminal receiver domain and a C-terminal output domain that binds DNA and interacts with sigma(70) to activate transcription of the Pho regulon. Phosphorylation of the receiver domain is required for activation of the protein. The mechanism of activation by phosphorylation has not yet been determined. To better understand the function of the receiver domain in controlling the activity of the output domain, a direct comparison was made between unphosphorylated PhoB and its solitary DNA-binding domain (PhoB(DBD)) for DNA binding and transcriptional activation. Using fluorescence anisotropy, it was found that PhoB(DBD) bound to the pho box with an affinity seven times greater than that of unphosphorylated PhoB. It was also found that PhoB(DBD) was better able to activate transcription than the full-length, unmodified protein. We conclude that the unphosphorylated receiver domain of PhoB silences the activity of its output domain. These results suggest that upon phosphorylation of the receiver domain of PhoB, the inhibition placed upon the output domain is relieved by a conformational change that alters interactions between the unphosphorylated receiver domain and the output domain.