National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R35 GM118130
米国
Life Sciences Research Foundation
Agouron Institute
米国
引用
ジャーナル: Nat Commun / 年: 2025 タイトル: Real-time capture of σ transcription initiation intermediates reveals mechanism of ATPase-driven activation by limited unfolding. 著者: Andreas U Mueller / Nina Molina / B Tracy Nixon / Seth A Darst / 要旨: 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 is unique among σ factors in ...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 is unique among σ factors in its structure and functional mechanism, requiring activation by specialized AAA+ ATPases. Eσ forms an inactive promoter complex where the N-terminal σ region I (σ-RI) threads through a small DNA bubble. On the opposite side of the DNA, the ATPase engages σ-RI within the pore of its hexameric ring. Here, we perform kinetics-guided structural analysis of de novo formed Eσ initiation complexes and engineer a biochemical assay to measure ATPase-mediated σ-RI translocation during promoter melting. We show that the ATPase exerts mechanical action to translocate about 30 residues of σ-RI through the DNA bubble, disrupting inhibitory structures of σ to allow full transcription bubble formation. A local charge switch of σ-RI from positive to negative may help facilitate disengagement of the otherwise processive ATPase, allowing subsequent σ disentanglement from the DNA bubble.
名称: dhsU (-60 to +30) non-template strand / タイプ: dna / ID: 7 詳細: Bases at the ends of the fragment (two positions on either side) were modified from the genomic sequence to G or C to stabilize the ends. コピー数: 1 / 分類: DNA
名称: dhsU (-60 to +30) template strand / タイプ: dna / ID: 8 詳細: Bases at the ends of the fragment (two positions on either side) were modified from the genomic sequence to G or C to stabilize the ends. コピー数: 1 / 分類: DNA
pH: 8 詳細: 40 mM Tris-HCl, pH 8/RT, 200 mM KCl, 10 mM MgCl2, 1 mM DTT; fluorinated fos-choline-8 (FC8F) added to a final concentration of 1.5 mM during grid preparation