Transcription from sigma54-dependent bacterial promoters can be regarded as a second paradigm for bacterial gene transcription. The initial sigma54-RNA polymerase (RNAP).promoter complex, the closed complex, is transcriptionally silent. The transcriptionally proficient sigma54-RNAP.promoter complex, the open complex, is formed upon remodeling of the closed complex by actions of a specialized activator protein that belongs to the AAA (ATPases associated with various cellular activities) protein family in an ATP hydrolysis-dependent reaction. The integrity of a highly conserved signature motif in the AAA activator (known as the GAFTGA motif) is important for the remodeling activity of the AAA activator and for open complex formation. We now provide evidence that the invariant threo-nine residue of the GAFTGA motif plays a role in sensing the DNA downstream of the sigma54-RNAP-binding site and in coupling this information to sigma54-RNAP via the conserved regulatory Region I domain of sigma54 during open complex formation.