We used bacteriophage T7-encoded transcription inhibitor gene protein 2 (gp2) as a probe to study the contribution of the Escherichia coli RNA polymerase (RNAP) β′ subunit jaw domain-the site of gp2 binding-to activator and ATP hydrolysis-dependent open complex formation by the σ54-RNAP. We show that, unlike σ70-dependent transcription, activated transcription by σS4-RNAP is resistant to gp2. In contrast, activator and ATP hydrolysis-independent transcription by σ54-RNAP is highly sensitive to gp2. We provide evidence that an activator- and ATP hydrolysis-dependent conformational change involving the β′ jaw domain and promoter DNA is the basis for gp2-resisiant transcription by σ54-RNAP. Our results establish that accessory factors bound to the upstream face of the RNAP, communicate with the β′ jaw domain, and that such communication is subjected to regulation.