Heterogeneous nucleotide occupancy stimulates functionality of phage shock protein F, an AAA+ transcriptional activator

The catalytic AAA+ domain (PspF1-275) of an enhancer-binding protein is necessary and sufficient to contact sigma54-RNA polymerase holoenzyme (Esigma54), remodel it, and in so doing catalyze open promoter complex formation. Whether ATP binding and hydrolysis is coordinated between subunits of PspF and the precise nature of the nucleotide(s) bound to the oligomeric forms responsible for substrate remodeling are unknown. We demonstrate that ADP stimulates the intrinsic ATPase activity of PspF1-275 and propose that this heterogeneous nucleotide occupancy in a PspF1-275 hexamer is functionally important for specific activity. Binding of ADP and ATP triggers the formation of functional PspF1-275 hexamers as shown by a gain of specific activity. Furthermore, ATP concentrations congruent with stoichiometric ATP binding to PspF1-275 inhibit ATP hydrolysis and Esigma54-promoter open complex formation. Demonstration of a heterogeneous nucleotide-bound state of a functional PspF1-275.Esigma54 complex provides clear biochemical evidence for heterogeneous nucleotide occupancy in this AAA+ protein. Based on our data, we propose a stochastic nucleotide binding and a coordinated hydrolysis mechanism in PspF1-275 hexamers.     

Cloning and characterization of the gene encoding RNA polymerase sigma factor sigma(54) of deep-sea piezophilic Shewanella violacea

We have recently reported that a sigma(54)-like factor recognizes a DNA element, designated as region A, upstream of a pressure-regulated operon in piezophilic Shewanella violacea strain DSS12 (Nakasone et al., FEMS Microbiology Lett. 176 (1999) 351-356). In this study, we isolated and characterized the rpoN gene of this piezophilic bacterium. The rpoN gene was found to encode a putative protein consisting of 492 amino acid residues with a predicted molecular mass of 55359 Da. Significant homology was evident comparing the rpoN sequence of S. violacea with that of Escherichia coli (62.8% identity), Vibrio anguillarum (61.7% identity) and Pseudomonas putida (57.0% identity). The DNA-binding domain at the C-terminus of sigma(54) is well conserved in the case of the S. violacea rpoN gene product and the helix-turn-helix motif and the RpoN box are also present. In addition, the conserved glutamine-rich domain is present at the N-terminus. sigma(54) in S. violacea was expressed at a relatively constant level under various growth conditions as determined by both primer extension and Western blotting analyses. By means of a recombinant plasmid, a hexahistidine-tagged derivative of the sigma(54) from strain DSS12 was overexpressed in Escherichia coli and purified to near homogeneity. An electrophoretic mobility shift assay demonstrated that the purified sigma(54) protein specifically recognizes region A in the above-mentioned pressure-regulated operon.