National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R35 GM118130
United States
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2021 Title: Structural origins of RNA polymerase open promoter complex stability. Authors: Ruth M Saecker / James Chen / Courtney E Chiu / Brandon Malone / Johanna Sotiris / Mark Ebrahim / Laura Y Yen / Edward T Eng / Seth A Darst / Abstract: The first step in gene expression in all organisms requires opening the DNA duplex to expose one strand for templated RNA synthesis. In , promoter DNA sequence fundamentally determines how fast the ...The first step in gene expression in all organisms requires opening the DNA duplex to expose one strand for templated RNA synthesis. In , promoter DNA sequence fundamentally determines how fast the RNA polymerase (RNAP) forms "open" complexes (RPo), whether RPo persists for seconds or hours, and how quickly RNAP transitions from initiation to elongation. These rates control promoter strength in vivo, but their structural origins remain largely unknown. Here, we use cryoelectron microscopy to determine the structures of RPo formed de novo at three promoters with widely differing lifetimes at 37 °C: λP (t ∼10 h), T7A1 (t ∼4 min), and a point mutant in λP (λP) (t ∼2 h). Two distinct RPo conformers are populated at λP, likely representing productive and unproductive forms of RPo observed in solution studies. We find that changes in the sequence and length of DNA in the transcription bubble just upstream of the start site (+1) globally alter the network of DNA-RNAP interactions, base stacking, and strand order in the single-stranded DNA of the transcription bubble; these differences propagate beyond the bubble to upstream and downstream DNA. After expanding the transcription bubble by one base (T7A1), the nontemplate strand "scrunches" inside the active site cleft; the template strand bulges outside the cleft at the upstream edge of the bubble. The structures illustrate how limited sequence changes trigger global alterations in the transcription bubble that modulate the RPo lifetime and affect the subsequent steps of the transcription cycle.
History
Deposition
Apr 23, 2021
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Header (metadata) release
Sep 29, 2021
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Map release
Sep 29, 2021
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Update
May 29, 2024
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Current status
May 29, 2024
Processing site: RCSB / Status: Released
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