Recent determinations of the structures of the bacterial RNA polymerase (RNAP) and promoter complex thereof establish that RNAP functions as a complex molecular machine that contains distinct structural modules that undergo major conformational changes during transcription. However, the contribution of the RNAP structural modules to transcription remains poorly understood. The bacterial core RNAP (α2ββ′ω; E) associates with a sigma (σ) subunit to form the holoenzyme (Eσ). A mutation removing the β subunit flap domain renders the Escherichia coli σ70 RNAP holoenzyme unable to recognize promoters. σ54 is the major variant σ subunit that utilizes enhancer-dependent promoters. Here, we determined the effects of β flap removal on σ54-dependent transcription. Our analysis shows that the role of the β flap in σ54-dependent and σ70-dependent transcription is different. Removal of the β flap does not prevent the recognition of σ54-dependent promoters, but causes multiple defects in σ54-dependent transcription. Most importantly, the β flap appears to orchestrate the proper formation of the Eσ54 regulatory center at the start site proximal promoter element where activator binds and DNA melting originates.