The protein sigma(54) associates with Escherichia coli core RNA polymerase to form a holoenzyme that binds promoters but is inactive in the absence of enhancer activation, Here, mutants of sigma(54) enabled polymerases to transcribe without enhancer protein and adenosine triphosphate. The mutations are in leucines within the NH2-terminal glutamine-rich domain of sigma(54). Multiple leucine substitutions mimicked the effect of enhancer protein, which suggests that the enhancer protein functions to disrupt a leucine patch. The results indicate that sigma(54) acts both as an inhibitor of polymerase activity and as a receptor that interacts with enhancer protein to overcome this inhibition, and that these two activities jointly confer enhancer responsiveness.