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

Abstract

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.