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

Andreas U. Mueller, Nina Molina, B. Tracy Nixon and Seth A. Darst ()
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Andreas U. Mueller: The Rockefeller University
Nina Molina: The Rockefeller University
B. Tracy Nixon: Penn State University
Seth A. Darst: The Rockefeller University

Nature Communications, 2025, vol. 16, issue 1, 1-18

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

Date: 2025
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DOI: 10.1038/s41467-025-61837-4

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