The prokaryotic activator protein NTRC binds to enhancer‐like elements and activates transcription in response to nitrogen limitation by catalysing open complex formation by sigma 54 RNA polymerase holoenzyme. Formation of open complexes requires the phosphorylated form of NTRC and the reaction is ATP dependent. We find that NTRC has an ATPase activity which is activated by phosphorylation and is strongly stimulated by the presence of DNA containing specific NTRC binding sites.

No abstract

Gene fusions in which the lac genes are under the control of each promoter in the Klebsiella pneumoniae, nitrogen fixation (nif) gene cluster have been constructed. These fusions have been used to examine positive control of the cluster and the response of individual genes to repression by ammonia and oxygen. De-repression of nif transcriptional units is coordinate and molybdate is required for maximal expression of the structural gene operon, which is autogenously regulated.

The enhancer-binding protein NIFA is required for transcriptional activation of nif promoters by the alternative holoenzyme form of RNA polymerase, which contains the sigma factor 54 ( N ). NIFA hydrolyzes nucleoside triphosphates to catalyze the isomerization of closed promoter complexes to transcriptionally competent open complexes. The activity of NIFA is antagonized by the regulatory protein NIFL in response to oxygen and fixed nitrogen in vivo. We have investigated the requirement for nucleotides in the formation and stability of open promoter complexes by NIFA and inhibition of its activity by NIFL at the Klebsiella pneumoniae nifH promoter. Open complexes formed by 54 -containing RNA polymerase are considerably more stable to heparin challenge in the presence of GTP than in the presence of ATP. This differential stability is most probably a consequence of GTP being the initiating nucleotide at this promoter. Adenosine nucleosides are specifically required for Azotobacter vinelandii NIFL to inhibit open complex formation by native NIFA, and the nucleoside triphosphatase activity of NIFA is strongly inhibited by NIFL under these conditions. We propose a model in which NIFL modulates the activity of NIFA via an adenosine nucleotide switch.A distinct mechanism of transcriptional activation is observed among the family of prokaryotic enhancer-binding proteins which interact with the holoenzyme form of RNA polymerase containing the alternative sigma factor 54 (E 54 ) (19,26). The nitrogen fixation regulatory protein NIFA is a member of this family which binds to upstream activator sequences (UAS) and catalyzes the isomerization of closed promoter complexes to the open complex in a reaction which requires hydrolysis of a nucleoside triphosphate (25). Productive interactions between NIFA and E 54 are enabled by DNA loop formation, which is facilitated by the binding of integration host factor (IHF) (18,30). The amino acid sequence of NIFA conforms to the three-domain model for the structure of pneumoniae NIFA activates transcription in the absence of specific DNA binding and possesses nucleoside triphosphatase activity (6). In contrast to its K. pneumoniae counterpart, the native Azotobacter vinelandii NIFA protein has been purified in a soluble form, and its properties with respect to DNA binding and catalysis of open complex formation have been characterized in vitro (2).In both K. pneumoniae and A. vinelandii, the activity of NIFA is controlled by a second regulatory protein, NIFL, in response to the environmental effectors oxygen and fixed nitrogen (7,22). Although NIFL proteins show homology in their C-terminal domains to the histidine protein kinase family of two-component regulatory proteins (14), NIFL and NIFA appear to interact at stoichiometric levels (5, 15), and phosphotransfer between the two proteins has not been detected in vitro (2, 21). Moreover, although A. vinelandii NIFL shows greater homology to the canonical histidine protein kinases than does K. pneumoniae NIFL and contains a conserved...

The bacterial activator protein NorR binds to enhancer-like elements, upstream of the promoter site, and activates σ54-dependent transcription of genes that encode nitric oxide detoxifying enzymes (NorVW), in response to NO stress. Unique to the norVW promoter in Escherichia coli is the presence of three enhancer sites associated with a binding site for σ54-RNA polymerase. Here we show that all three sites are required for NorR-dependent catalysis of open complex formation by σ54-RNAP holoenzyme (Eσ54). We demonstrate that this is essentially due to the need for all three enhancers for maximal ATPase activity of NorR, energy from which is used to remodel the closed Eσ54 complex and allow melting of the promoter DNA. We also find that site-specific DNA binding per se promotes oligomerisation but the DNA flanking the three sites is needed to further stabilise the functional higher order oligomer of NorR at the enhancers.