Journal: Elife / Year: 2019 Title: Crl activates transcription by stabilizing active conformation of the master stress transcription initiation factor. Authors: Juncao Xu / Kaijie Cui / Liqiang Shen / Jing Shi / Lingting Li / Linlin You / Chengli Fang / Guoping Zhao / Yu Feng / Bei Yang / Yu Zhang / Abstract: σ is a master transcription initiation factor that protects bacterial cells from various harmful environmental stresses including antibiotic pressure. Although its mechanism remains unclear, it is ...σ is a master transcription initiation factor that protects bacterial cells from various harmful environmental stresses including antibiotic pressure. Although its mechanism remains unclear, it is known that full activation of σ-mediated transcription requires a σ-specific activator, Crl. In this study, we determined a 3.80 Å cryo-EM structure of an transcription activation complex ( Crl-TAC) comprising σ-RNA polymerase (σ-RNAP) holoenzyme, Crl, and a nucleic-acid scaffold. The structure reveals that Crl interacts with domain 2 of σ (σ) and the RNAP core enzyme, but does not contact promoter DNA. Results from subsequent hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicate that Crl stabilizes key structural motifs within σ to promote the assembly of the σ-RNAP holoenzyme and also to facilitate formation of an RNA polymerase-promoter DNA open complex (RPo). Our study demonstrates a unique DNA contact-independent mechanism of transcription activation, thereby defining a previously unrecognized mode of transcription activation in cells.
History
Deposition
Jul 21, 2019
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Header (metadata) release
Jan 1, 2020
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Map release
Jan 1, 2020
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Update
Mar 27, 2024
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Current status
Mar 27, 2024
Processing site: PDBj / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Name: RNA (5'-R(*CP*UP*CP*GP*A)-3') / type: rna / ID: 8 / Number of copies: 1
Source (natural)
Organism: synthetic construct (others)
Molecular weight
Theoretical: 1.545984 KDa
Sequence
String:
CUCGA
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Macromolecule #10: ZINC ION
Macromolecule
Name: ZINC ION / type: ligand / ID: 10 / Number of copies: 2 / Formula: ZN
Molecular weight
Theoretical: 65.409 Da
+
Macromolecule #11: MAGNESIUM ION
Macromolecule
Name: MAGNESIUM ION / type: ligand / ID: 11 / Number of copies: 1 / Formula: MG
Molecular weight
Theoretical: 24.305 Da
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Concentration
12 mg/mL
Buffer
pH: 7.5 Component:
Concentration
Formula
Name
10.0 mM
Hepes
Hepes
50.0 mM
KCl
Potassium Chloride
5.0 mM
MgCl2
Magnesium Chloride
3.0 mM
DTT
DL-Dithiothreitol
Grid
Model: C-flat-1.2/1.3 4C / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 120 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: OTHER
Vitrification
Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 8 seconds before plunging.
Details
This sample was monodisperse
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 3290 / Average exposure time: 8.0 sec. / Average electron dose: 1.675 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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