Expression from the Escherichia coli nir promoter is co-dependent on both the FNR protein (an anaerobically triggered transcription activator) and NarL or NarP proteins (transcription activators triggered by nitrite and nitrate). We found previously that FNR binds to a site centred at position - 41.5 at the nir promoter, but that FNR-dependent activation is repressed by IHF binding to a site centred at position -88 (IHF I) and Fis binding to sites centred at positions -142 (Fis I) and +23 (Fis II). Here, we have studied the binding of purified IHF, Fis and FNR to the nir promoter in vitro. Our results show that the nir promoter contains a second IHF site at position -115 (IHF II) and a third Fis site at position -97 (Fis III), and that IHF, Fis and FNR can bind together to form multiprotein complexes. Surprisingly, IHF binding at the IHF II site increases FNR-dependent activation by decreasing the repression mediated by IHF and Fis binding at the other sites. In previous work, we found that NarL or NarP activates the nir promoter by binding to a site centred at position -69.5 and counteracting the repressive effects of IHF and Fis. We now show that NarL can displace IHF bound at the IHF I site, but IHF is unable to displace bound NarL. We suggest that NarL interferes with IHF binding at the nir promoter by distorting the minor groove at its target site, and we argue that the resulting activation by NarL results from remodelling of the local nucleoprotein structure to facilitate FNR-dependent transcription.
- DNA-Binding Proteins/physiology
- Escherichia coli
- Escherichia coli Proteins/physiology
- Gene Expression Regulation, Bacterial
- Iron-Sulfur Proteins/metabolism
- Promoter Regions, Genetic