In vitro roles of invariant helix-turn-helix motif residue R383 in sigma(54) (sigma(N)).
In vitro DNA-binding and transcription properties of sigma(54) proteins with the invariant Arg383 in the putative helix-turn-helix motif of the DNA-binding domain substituted by lysine or alanine are described. We show that R383 contributes to maintaining stable holoenzyme-promoter complexes in which limited DNA opening downstream of the -12 GC element has occurred. Unlike wild-type sigma(54), holoenzymes assembled with the R383A or R383K mutants could not form activator-independent, heparin-stable complexes on heteroduplex Sinorhizobium meliloti nifH DNA mismatched next to the GC. Using longer sequences of heteroduplex DNA, heparin-stable complexes formed with the R383K and, to a lesser extent, R383A mutant holoenzymes, but only when the activator and a hydrolysable nucleotide was added and the DNA was opened to include the -1 site. Although R383 appears inessential for polymerase isomerisation, it makes a significant contribution to maintaining the holoenzyme in a stable complex when melting is initiating next to the GC element. Strikingly, Cys383-tethered FeBABE footprinting of promoter DNA strongly suggests that R383 is not proximal to promoter DNA in the closed complex. This indicates that R383 is not part of the regulatory centre in the sigma(54) holoenzyme, which includes the -12 promoter region elements. R383 contributes to several properties, including core RNA polymerase binding and to the in vivo stability of sigma(54).
Department of Biology, Imperial College of Science, Technology and Medicine, Sir Alexander Fleming Building, Imperial College Road, London SW7 2AZ, UK.
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Wigneshweraraj
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Ishihama
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Buck
D001120:Arginine / Q000235:genetics; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D015870:Gene Expression; D019077:Helix-Turn-Helix Motifs / Q000235:genetics*; D007711:Klebsiella pneumoniae / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism*; D016962:Sinorhizobium meliloti / Q000235:genetics
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012808:Sigma Factor; C497751:rpoN protein, E coli; D001120:Arginine; D010088:Oxidoreductases; C029130:nitrogenase reductase; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54