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).