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: 2023 Title: A general mechanism for transcription bubble nucleation in bacteria. Authors: Andreas U Mueller / James Chen / Mengyu Wu / Courtney Chiu / B Tracy Nixon / Elizabeth A Campbell / Seth A Darst / Abstract: Bacterial transcription initiation requires σ factors for nucleation of the transcription bubble. The canonical housekeeping σ factor, σ, nucleates DNA melting via recognition of conserved bases ...Bacterial transcription initiation requires σ factors for nucleation of the transcription bubble. The canonical housekeeping σ factor, σ, nucleates DNA melting via recognition of conserved bases of the promoter -10 motif, which are unstacked and captured in pockets of σ. By contrast, the mechanism of transcription bubble nucleation and formation during the unrelated σ-mediated transcription initiation is poorly understood. Herein, we combine structural and biochemical approaches to establish that σ, like σ, captures a flipped, unstacked base in a pocket formed between its N-terminal region I (RI) and extra-long helix features. Strikingly, RI inserts into the nascent bubble to stabilize the nucleated bubble prior to engagement of the obligate ATPase activator. Our data suggest a general paradigm of transcription initiation that requires σ factors to nucleate an early melted intermediate prior to productive RNA synthesis.
Model: C-flat-1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV Details: Octyl beta-D-glucopyranoside (beta-OG) was added to the sample to 0.1%w/v final concentration (from 10x stock) just prior to plunge vitrification.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recording
Film or detector model: GATAN K3 (6k x 4k) / Number real images: 12294 / Average exposure time: 2.0 sec. / Average electron dose: 51.0 e/Å2 Details: dose-fractionation with 0.05 seconds per frame (=40 frames)
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
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Image processing
Startup model
Type of model: OTHER / Details: ab initio generated map
Initial angle assignment
Type: MAXIMUM LIKELIHOOD
Final angle assignment
Type: MAXIMUM LIKELIHOOD
Final reconstruction
Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: PHENIX / Software - details: phenix.combine_focused_maps Details: Particle numbers and FSC resolution estimate of the full map given Number images used: 602287
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