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 the two-component regulatory system consisting of the two proteins EnvZ and OmpR. EnvZ is an integral inner membrane protein that is phosphorylated by cytoplasmic ATP on a histidine residue. EnvZ modulates the activity of OmpR by phosphorylation and dephosphorylation. Phospho-OmpR (OmpR-P) binds to the porin genes ompF and ompC to regulate their expression. The simple affinity model predicts that as the concentration of OmpR-P increases, initially high-affinity binding sites on ompF are filled. Then binding sites of lower affinity on ompF and ompC are occupied and this ordered binding accounts for the differential expression of the porin genes. We demonstrate that acetyl phosphate phosphorylates OmpR at aspartate 55, the same residue phosphorylated by the kinase EnvZ. Quantification of the level of OmpR-P by HPLC and direct measurement 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 not sufficiently different to provide a strong basis for discrimination. The consequences of these observations for the simple affinity model are considered.
Department of Molecular Microbiology & Immunology L220, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR, 97201, USA.
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D001425:Bacterial Outer Membrane Proteins / Q000502:physiology; D001426:Bacterial Proteins / Q000502:physiology; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004268:DNA-Binding Proteins / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005809:Genes, Regulator / Q000235:genetics*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D009154:Mutation; D010755:Organophosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000502:physiology; D010766:Phosphorylation; D018272:Porins / Q000235:genetics*; D016415:Sequence Alignment; D015534:Trans-Activators / Q000502:physiology*; D015533:Transcriptional Activation / Q000502:physiology
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D010755:Organophosphates; D010750:Phosphoproteins; D018272:Porins; D015534:Trans-Activators; C112177:osmolarity response regulator proteins; C011632:acetyl phosphate; C057843:envZ protein, E coli