Journal: Nucleic Acids Res / Year: 2019 Title: Structures and mechanism of transcription initiation by bacterial ECF factors. Authors: Chengli Fang / Lingting Li / Liqiang Shen / Jing Shi / Sheng Wang / Yu Feng / Yu Zhang / Abstract: Bacterial RNA polymerase (RNAP) forms distinct holoenzymes with extra-cytoplasmic function (ECF) σ factors to initiate specific gene expression programs. In this study, we report a cryo-EM structure ...Bacterial RNA polymerase (RNAP) forms distinct holoenzymes with extra-cytoplasmic function (ECF) σ factors to initiate specific gene expression programs. In this study, we report a cryo-EM structure at 4.0 Å of Escherichia coli transcription initiation complex comprising σE-the most-studied bacterial ECF σ factor (Ec σE-RPo), and a crystal structure at 3.1 Å of Mycobacterium tuberculosis transcription initiation complex with a chimeric σH/E (Mtb σH/E-RPo). The structure of Ec σE-RPo reveals key interactions essential for assembly of E. coli σE-RNAP holoenzyme and for promoter recognition and unwinding by E. coli σE. Moreover, both structures show that the non-conserved linkers (σ2/σ4 linker) of the two ECF σ factors are inserted into the active-center cleft and exit through the RNA-exit channel. We performed secondary-structure prediction of 27,670 ECF σ factors and find that their non-conserved linkers probably reach into and exit from RNAP active-center cleft in a similar manner. Further biochemical results suggest that such σ2/σ4 linker plays an important role in RPo formation, abortive production and promoter escape during ECF σ factors-mediated transcription initiation.
History
Deposition
Jan 26, 2019
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Header (metadata) release
May 29, 2019
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Map release
May 29, 2019
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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 #9: MAGNESIUM ION
Macromolecule
Name: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 1 / Formula: MG
Molecular weight
Theoretical: 24.305 Da
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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
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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
15 mg/mL
Buffer
pH: 7.5 Component:
Concentration
Formula
Name
10.0 mmol/L
C8H18N2O4S
HEPES
50.0 mmol/L
KCl
Potassium Chloride
5.0 mmol/L
MgCl2
magnesium chloride
3.0 mmol/L
C4H10O2S2
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: 282.65 K / Instrument: FEI VITROBOT MARK III
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number grids imaged: 1 / Number real images: 2089 / Average exposure time: 8.0 sec. / Average electron dose: 1.665 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stage
Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
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Image processing
Particle selection
Number selected: 503749
Startup model
Type of model: NONE
Final reconstruction
Number classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.02 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 195058
Initial angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final 3D classification
Number classes: 4 / Software - Name: RELION (ver. 2.1)
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