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

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