The structures of the bacterial RNA polymerase holoenzyme have provided detailed information about the intersubunit interactions within the holoenzyme. Functional analysis indicates that one of these is critical in enabling the holoenzyme to recognize the major class of bacterial promoters. It has been suggested that this interaction, involving the flap domain of the β subunit and conserved region 4 of the σ subunit, is a potential target for regulation. Here we provide genetic and biochemical evidence that the σ region 4/β-flap interaction is targeted by the transcription factor AsiA. Specifically, we show that AsiA competes directly with the β-flap for binding to σ region 4, thereby inhibiting transcription initiation by disrupting the σ region 4/β-flap interaction.