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

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年8月4日

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  • Andreas U. Mueller,
  • Nina Molina,
  • B. Tracy Nixon,
  • Seth A. Darst

摘要

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.