The sigma(54) promoter specificity factor is distinct from other bacterial RNA polymerase (RNAP) sigma factors in that it forms a transcriptionally silent closed complex upon promoter binding. Transcriptional activation occurs through a nucleotide-dependent isomerization of sigma(54), mediated via its interactions with an enhancer-binding activator protein that utilizes the energy released in ATP hydrolysis to effect structural changes in sigma(54) and core RNA polymerase. The organization of sigma(54)-promoter and sigma(54)-RNAP-promoter complexes was investigated by fluorescence resonance energy transfer assays using sigma(54) single cysteine-mutants labeled with an acceptor fluorophore and donor fluorophore-labeled DNA sequences containing mismatches that mimic nifH early- and late-melted promoters. The results show that sigma(54) undergoes spatial rearrangements of functionally important domains upon closed complex formation. sigma(54) and sigma(54)-RNAP promoter complexes reconstituted with the different mismatched DNA constructs were assayed by the addition of the activator phage shock protein F in the presence or absence of ATP and of non-hydrolysable analogues. Nucleotide-dependent alterations in fluorescence resonance energy transfer efficiencies identify different functional states of the activator-sigma(54)-RNAP-promoter complex that exist throughout the mechano-chemical transduction pathway of transcriptional activation, i.e. from closed to open promoter complexes. The results suggest that open complex formation only occurs efficiently on replacement of a repressive fork junction with down-stream melted DNA.