Stable accumulation of sigma54 in Helicobacter pylori requires the novel protein HP0958

Several flagellar genes in Helicobacter pylori are dependent on sigma(54) (RpoN) for their expression. These genes encode components of the basal body, the hook protein, and a minor flagellin, FlaB. A protein-protein interaction map for H. pylori constructed from a high-throughput screen of a yeast two-hybrid assay (http://pim.hybrigenics.com/pimriderext/common/) revealed interactions between sigma(54) and the conserved hypothetical protein HP0958. To see if HP0958 influences sigma(54) function, the corresponding gene was disrupted with a kanamycin resistance gene (aphA3) in H. pylori ATCC 43504 and the resulting mutant was analyzed. The hp0958:aphA3 mutant was nonmotile and failed to produce flagella. Introduction of a functional copy of hp0958 into the genome of the hp0958:aphA3 mutant restored flagellar biogenesis and motility. The hp0958:aphA3 mutant was deficient in expressing two sigma(54)-dependent reporter genes, flaB'-'xylE and hp1120'-'xylE. Levels of sigma(54) in the hp0958 mutant were substantially lower than those in the parental strain, suggesting that the failure of the mutant to express the genes in the RpoN regulon and produce flagella was due to reduced sigma(54) levels. Expressing sigma(54) at high levels by putting rpoN under the control of the ureA promoter restored flagellar biogenesis and motility in the hp0958:aphA3 mutant. Turnover of sigma(54) was more rapid in the hp0958:aphA3 mutant than it was in the wild-type strain, suggesting that HP0958 supports wild-type sigma(54) levels in H. pylori by protecting it from proteolysis.     

Active recruitment of sigma54-RNA polymerase to the Pu promoter of Pseudomonas putida: role of IHF and alphaCTD.

The sequence elements determining the binding of the sigma54-containing RNA polymerase (sigma54-RNAP) to the Pu promoter of Pseudomonas putida have been examined. Contrary to previous results in related systems, we show that the integration host factor (IHF) binding stimulates the recruitment of the enzyme to the -12/-24 sequence motifs. Such a recruitment, which is fully independent of the activator of the system, XylR, requires the interaction of the C-terminal domain of the alpha subunit of RNAP with specific DNA sequences upstream of the IHF site which are reminiscent of the UP elements in sigma70 promoters. Our data show that this interaction is mainly brought about by the distinct geometry of the promoter region caused by IHF binding and the ensuing DNA bending. These results support the view that binding of sigma54-RNAP to a promoter is a step that can be subjected to regulation by factors (e.g. IHF) other than the sole intrinsic affinity of sigma54-RNAP for the -12/-24 site.     

Binding affinity of Escherichia coli RNA polymerase*sigma54 holoenzyme for the glnAp2, nifH and nifL promoters

Escherichia coli RNA polymerase associated with the sigma54 factor (RNAP*sigma54) is a holoenzyme form that transcribes a special class of promoters not recognized by the standard RNA polymerase*sigma70 com plex. Promoters for RNAP*sigma54 vary in their overall 'strength' and show differences in their response to the presence of DNA curvature between enhancer and promoter. In order to examine whether these effects are related to the promoter affinity, we have determined the equilibrium dissociation constant K(d) for the binding of RNAP*sigma54 to the three promoters glnAp2, nifH and nifL. Binding studies were conducted by monitoring the changes in fluorescence anisotropy upon titrating RNAP*sigma54 to carboxyrhodamine-labeled DNA duplexes. For the glnAp2 and nifH promoters similar values of K(d) = 0.94 +/- 0.55 nM and K(d) = 0.85 +/- 0.30 nM were determined at physiological ionic strength, while the nifL promoter displayed a significantly weaker affinity with K(d) = 8.5 +/- 1.9 nM. The logarithmic dependence of K(d) on the ionic strength I was -Deltalog(K(d))/Deltalog(I) = 6.1 +/- 0.5 for the glnAp2, 5.2 +/- 1.2 for the nifH and 2.1 +/- 0.1 for the nifL promoter. This suggests that the polymerase can form fewer ion pairs with the nifL promoter, which would account for its weaker binding affinity.     

Recruitment of sigma54-RNA polymerase to the Pu promoter of Pseudomonas putida through integration host factor-mediated positioning switch of alpha subunit carboxyl-terminal domain on an UP-like element

The interactions between the sigma54-containing RNA polymerase (sigma54-RNAP) and the region of the Pseudomonas putida Pu promoter spanning from the enhancer to the binding site for the integration host factor (IHF) were analyzed both by DNase I and hydroxyl radical footprinting. A short Pu region centered at position -104 was found to be involved in the interaction with sigma54-RNAP, both in the absence and in the presence of IHF protein. Deletion or scrambling of the -104 region strongly reduced promoter affinity in vitro and promoter activity in vivo, respectively. The reduction in promoter affinity coincided with the loss of IHF-mediated recruitment of the sigma54-RNAP in vitro. The experiments with oriented-alpha sigma54-RNAP derivatives containing bound chemical nuclease revealed interchangeable positioning of only one of the two alpha subunit carboxyl-terminal domains (alphaCTDs) both at the -104 region and in the surroundings of position -78. The addition of IHF resulted in perfect position symmetry of the two alphaCTDs. These results indicate that, in the absence of IHF, the sigma54-RNAP asymmetrically uses only one alphaCTD subunit to establish productive contacts with upstream sequences of the Pu promoter. In the presence of IHF-induced curvature, the closer proximity of the upstream DNA to the body of the sigma54-RNAP can allow the other alphaCTD to be engaged in and thus favor closed complex formation.     

Nucleotide-dependent conformational changes in the sigma54-dependent activator DctD

Activators of sigma(54)-RNA polymerase holoenzyme couple ATP hydrolysis to formation of an open promoter complex. DctD(Delta1-142), a truncated and constitutively active form of the sigma(54)-dependent activator DctD from Sinorhizobium meliloti, displayed an altered DNase I footprint at its binding site located upstream of the dctA promoter in the presence of ATP. The altered footprint was not observed for a mutant protein with a substitution at or near the putative arginine finger, a conserved arginine residue thought to contact the nucleotide. These data suggest that structural changes in DctD(Delta1-142) during ATP hydrolysis can be detected by alterations in the DNase I footprint of the protein and may be communicated by interactions between bound nucleotide and the arginine finger. In addition, kinetic data for changes in fluorescence energy transfer upon binding of 2'(3')-O-(N-methylanthraniloyl)-ATP (Mant-ATP) to DctD(Delta1-142) and DctD suggested that these proteins undergo multiple conformational changes following ATP binding.