Structural insights into the activity of enhancer-binding proteins

Activators of bacterial sigma(54)-RNA polymerase holoenzyme are mechanochemical proteins that use adenosine triphosphate (ATP) hydrolysis to activate transcription. We have determined by cryogenic electron microscopy (cryo-EM) a 20 angstrom resolution structure of an activator, phage shock protein IF [PspF((1-275))], which is bound to an ATP transition state analog in complex with its basal factor, sigma(54). By fitting the crystal structure of PspF((1-275)) at 1.75 angstroms into the EM map, we identified two loops involved in binding sigma(54). Comparing enhancer-binding structures in different nucleotide states and mutational analysis led us to propose nucleotide-dependent conformational changes that free the loops for association with sigma(54)..