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bioRxiv · 10.1101/2025.02.07.637174

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

Mueller, A. U.·Molina, N.·Darst, S. A.
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Abstract

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.

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Mueller, A. U., Molina, N., Darst, S. A.. 2025-02-08. Real-time capture of σN transcription initiation intermediates reveals mechanism of ATPase-driven activation by limited unfolding. https://doi.org/10.1101/2025.02.07.637174

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