Suppression of FNR-dependent transcription activation at the Escherichia coli nir promoter by Fis, IHF and H-NS: modulation of transcription initiation by a complex nucleo-protein assembly

Expression from the Escherichia coil nir promoter is co-dependent on both t he FNR protein (an anaerobically triggered transcription activator) and the NarL or Harp proteins (transcription activators triggered by nitrite and n itrate). Under anaerobic conditions, FNR binds to a site centred between po sitions -41 and -42, activating transcription of the nir operon. In previou s work, we showed that this activation is suppressed by the binding of Fis protein, and at least one other protein, to sequence elements located upstr eam of the nir promoter. We proposed that the binding of NarL or Harp to a site centred between positions -69 and -70 counteracts this suppression, re sulting in increased transcription in response to nitrite or nitrate. Here we have further investigated the different proteins that downregulate the n ir promoter. We show that the nir promoter is repressed by three DNA bindin g proteins, Fis, IHF and H-NS. We demonstrate that, in addition to binding to its previously characterized upstream site located at position -142, Fis also binds to a second downstream site located at position +23. A second s uppressing factor is IHF, that binds to a site located at position -88. Fin ally, the nucleoid associated protein, H-NS, preferentially binds to upstre am sequences at the nir promoter and represses promoter activity. The assoc iation of Fis, IHF and H-NS suggests that nir promoter DNA is sequestrated into a highly ordered nucleo-protein structure that represses FNR-dependent transcription activation. NarL and NarP can relieve both IHF- and Fis-medi ated repression, but are unable to counteract H-NS-mediated repression.