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.