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J. Bacteriol., 05 1995, 2490-2496, Vol 177, No. 9
Copyright © 1995, American Society for Microbiology

Active contribution of two domains to cooperative DNA binding of the enhancer-binding protein nitrogen regulator I (NtrC) of Escherichia coli: stimulation by phosphorylation and the binding of ATP

P Chen and LJ Reitzer
Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson 75083-0688, USA.

Activation by the prokaryotic activator nitrogen regulator I (NRI, or NtrC) of Escherichia coli requires an interaction between two NRI dimers. ATP-dependent phosphorylation stimulates this tetramerization, which can be detected as cooperative binding to DNA. A polypeptide containing only the DNA-binding carboxyl-terminal domain has been previously shown to bind noncooperatively to DNA. Our primary purpose was to determine whether the highly conserved N-terminal domain or the ATP-binding central domain is required for cooperative DNA binding. Because ATP was present in the experiments that showed that phosphorylation enhances cooperative bindings, it is possible that ATP and not phosphorylation stimulated cooperative binding. Our secondary purpose was to separately assess the effects of ATP and phosphorylation on cooperative binding. We showed that a variant with a deletion of the central domain, NRI-(delta 143-398), binds cooperatively as well as unphosphorylated wild-type NRI, implying that the N-terminal domain mediates phosphorylation-independent cooperative binding. Phosphorylation of NRI-(delta 143-398) did not further stimulate this binding, suggesting that the ATP-binding central domain may be required for the phosphorylation-dependent enhancement. Cooperative binding was enhanced by either acetyl-phosphate-dependent (i.e., ATP-independent) phosphorylation of NRI or the specific binding of ATP to the central domain. Their effects were not additive, a finding which is consistent with the interpretation that each promotes a similar dimer-dimer interaction. We discuss these results within the context of the hypothesis that the highly conserved N-terminal domain mediates phosphorylation-independent cooperativity and the central domain is required for cooperativity stimulated by ATP binding or phosphorylation.

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