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

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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)

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.

Suggested Citation

  • Andreas U. Mueller & Nina Molina & B. Tracy Nixon & Seth A. Darst, 2025. "Real-time capture of σN transcription initiation intermediates reveals mechanism of ATPase-driven activation by limited unfolding," Nature Communications, Nature, vol. 16(1), pages 1-18, December.
    • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-61837-4
    DOI: 10.1038/s41467-025-61837-4
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    References listed on IDEAS

    as
    1. Mikhail Kavalchuk & Ahmad Jomaa & Andreas U. Müller & Eilika Weber-Ban, 2022. "Structural basis of prokaryotic ubiquitin-like protein engagement and translocation by the mycobacterial Mpa-proteasome complex," Nature Communications, Nature, vol. 13(1), pages 1-13, December.
    2. Claudio Alfieri & Leifu Chang & David Barford, 2018. "Mechanism for remodelling of the cell cycle checkpoint protein MAD2 by the ATPase TRIP13," Nature, Nature, vol. 559(7713), pages 274-278, July.
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