The sigma 54 promoter specificity factor is distinct from sigma 70-type factors. The sigma 54-RNA polymerase binds to promoters with conserved sequence elements at -24 and -12 and utilizes specialized enhancer-binding activators to convert, through an ATP-dependent process, closed promoter complexes to open promoter complexes. The interface between sigma 54-RNA polymerase and promoter DNA is poorly characterized, contrasting with sigma 70. Here, sigma 54 was modified with strategically positioned cleavage reagents to provide physical evidence that the highly conserved RpoN box motif of sigma 54 is close to and may therefore interact with the consensus -24 promoter element. We show that the spatial relationship between the sigma 54-RNA polymerase and the -24 promoter element remains unchanged during closed to open complex conversion and transcription initiation but changes during the early elongation phase. In contrast, the spatial relationship between sigma 54-RNA polymerase and the consensus -12 promoter element changes upon conversion of the closed promoter complex to an open one. We provide evidence that some -12 promoter region-sigma 54 interactions are dependent upon either the core RNA polymerase or a fork junction DNA structure at the -12-position, indicating that DNA fork junctions can substitute for core RNAP. We also show the beta-subunit flap domain contributes to different sets of sigma-promoter DNA interactions at sigma 54- and sigma 70-dependent promoters.