RELATIVE BINDING AFFINITIES OF OMPR AND OMPR-PHOSPHATE AT THE OMPF AND OMPC REGULATORY SITES

In Escherichia coli, porin gene expression is regulated, in part, by t he two-component regulatory system consisting of the two proteins EnvZ and OmpR. EnvZ is an integral inner membrane protein that is phosphor ylated by cytoplasmic Am on a histidine residue. EnvZ modulates the ac tivity of OmpR by phosphorylation and dephosphorylation. Phospho-OmpR (OmpR-P) binds to the porin genes ompF and ompC to regulate their expr ession. The simple affinity model predicts that as the concentration o f OmpR-P increases, initially high-affinity binding sites on ompF are filled. Then binding sites of lower affinity on ompF and ompC are occu pied and this ordered binding accounts for the differential expression of the porin genes. We demonstrate that acetyl phosphate phosphorylat es OmpR at aspartate 55, the same residue phosphorylated by the kinase EnvZ. Quantification of the level of OmpR-P by HPLC and direct measur ement of the binding affinities enabled us to test the affinity model. Our results indicate that phosphorylation dramatically increases the affinity of OmpR for its binding sites (greater than tenfold). We also show that the affinities of OmpR-P for F1 and C1 binding sites are no t sufficiently different to provide a strong basis for discrimination. The consequences of these observations for the simple affinity model are considered. (C) 1998 Academic Press.