Real-time capture of σN transcription initiation intermediates reveals mechanism of ATPase-driven activation by limited unfolding

Bacterial {sigma} factors bind RNA polymerase (E) to form holoenzyme (E{sigma}), conferring promoter specificity to E and playing a key role in transcription bubble formation. {sigma}N is unique among {sigma} factors in its structure and functional mechanism, requiring activation by specialized AAA+ ATPases. E{sigma}N forms an inactive promoter complex where the N-terminal {sigma}N region I ({sigma}N-RI) threads through a small DNA bubble. On the opposite side of the DNA, the ATPase engages {sigma}N-RI within the pore of its hexameric ring. Here, we perform kinetics-guided structural analysis of de novo formed E{sigma}N initiation complexes and engineer a biochemical assay to measure ATPase-mediated {sigma}N-RI translocation during promoter melting. We show that the ATPase exerts mechanical action to translocate about 30 residues of {sigma}N-RI through the DNA bubble, disrupting inhibitory structures of {sigma}N to allow full transcription bubble formation. A local charge switch of {sigma}N-RI from positive to negative may help facilitate disengagement of the otherwise processive ATPase, allowing subsequent {sigma}N disentanglement from the DNA bubble.