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- EMDB-22247: Cryo-EM structure of E. coli RNAP-promoter initial transcribing c... -

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Basic information

Entry
Database: EMDB / ID: EMD-22247
TitleCryo-EM structure of E. coli RNAP-promoter initial transcribing complex with 5-nt RNA transcript (RPitc-5nt)
Map data
SampleRNAP-promoter initial transcribing complex with 5-nt RNA transcript (RPitc-5nt)
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • RNA polymerase sigma factor RpoD
  • (nucleic-acidNucleic acid) x 3
  • (ligand) x 3
Function / homology
Function and homology information


DNA-templated transcription, initiation => GO:0006352 / sigma factor activity / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat / transcription, DNA-templated / protein dimerization activity / magnesium ion binding / DNA binding ...DNA-templated transcription, initiation => GO:0006352 / sigma factor activity / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat / transcription, DNA-templated / protein dimerization activity / magnesium ion binding / DNA binding / zinc ion binding / cytosol
Similarity search - Function
Sigma-70, non-essential region / RNA polymerase sigma factor 70, non-essential domain / RNA polymerase sigma factor 70, region 1.1 / Sigma-70 factor, region 1.1 superfamily / Sigma-70 factor, region 1.1 / Sigma-70 factors family signature 1. / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / RNA polymerase sigma-70 region 1.2 / Sigma-70 factor, region 1.2 ...Sigma-70, non-essential region / RNA polymerase sigma factor 70, non-essential domain / RNA polymerase sigma factor 70, region 1.1 / Sigma-70 factor, region 1.1 superfamily / Sigma-70 factor, region 1.1 / Sigma-70 factors family signature 1. / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / RNA polymerase sigma-70 region 1.2 / Sigma-70 factor, region 1.2 / Sigma-70 factors family signature 2. / Sigma-70 region 3 / RNA polymerase sigma-70 region 3 / RNA polymerase sigma-70 / Sigma-70, region 4 / RNA polymerase sigma-70 region 4 / Sigma-70 region 2 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 like domain / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like / DNA-directed RNA polymerase, omega subunit / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / RNA polymerase, alpha subunit, C-terminal / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / RNA polymerase beta subunit external 1 domain / DNA-directed RNA polymerase, beta subunit, external 1 domain / DNA-directed RNA polymerase beta subunit, bacterial-type / DNA-directed RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb6 / RPB6/omega subunit-like superfamily / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb2, domain 2 superfamily / : / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 4 / RNA polymerase, N-terminal / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 5 / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 5 / RNA polymerase, alpha subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase beta subunit / RNA polymerase, beta subunit, protrusion / RNA polymerase Rpb2, domain 2 / RNA polymerases D / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase Rpb3/RpoA insert domain / DNA-directed RNA polymerase, insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, OB-fold / RNA polymerases beta chain signature. / RNA polymerase Rpb2, domain 6 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta / RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta'
Similarity search - Component
Biological speciesEscherichia coli O157:H7 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsYang Y / Liu C / Liu B
CitationJournal: Nat Commun / Year: 2021
Title: Structural visualization of transcription activated by a multidrug-sensing MerR family regulator.
Authors: Yang Yang / Chang Liu / Wei Zhou / Wei Shi / Ming Chen / Baoyue Zhang / David G Schatz / Yangbo Hu / Bin Liu /
Abstract: Bacterial RNA polymerase (RNAP) holoenzyme initiates transcription by recognizing the conserved -35 and -10 promoter elements that are optimally separated by a 17-bp spacer. The MerR family of ...Bacterial RNA polymerase (RNAP) holoenzyme initiates transcription by recognizing the conserved -35 and -10 promoter elements that are optimally separated by a 17-bp spacer. The MerR family of transcriptional regulators activate suboptimal 19-20 bp spacer promoters in response to myriad cellular signals, ranging from heavy metals to drug-like compounds. The regulation of transcription by MerR family regulators is not fully understood. Here we report one crystal structure of a multidrug-sensing MerR family regulator EcmrR and nine cryo-electron microscopy structures that capture the EcmrR-dependent transcription process from promoter opening to initial transcription to RNA elongation. These structures reveal that EcmrR is a dual ligand-binding factor that reshapes the suboptimal 19-bp spacer DNA to enable optimal promoter recognition, sustains promoter remodeling to stabilize initial transcribing complexes, and finally dissociates from the promoter to reverse DNA remodeling and facilitate the transition to elongation. Our findings yield a comprehensive model for transcription regulation by MerR family factors and provide insights into the transition from transcription initiation to elongation.
History
DepositionJun 28, 2020-
Header (metadata) releaseApr 7, 2021-
Map releaseApr 7, 2021-
UpdateMay 26, 2021-
Current statusMay 26, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.7
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.7
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6xll
  • Surface level: 0.7
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_22247.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.33 Å/pix.
x 256 pix.
= 341.248 Å
1.33 Å/pix.
x 256 pix.
= 341.248 Å
1.33 Å/pix.
x 256 pix.
= 341.248 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.333 Å
Density
Contour LevelBy AUTHOR: 0.7 / Movie #1: 0.7
Minimum - Maximum-4.073657 - 6.300571
Average (Standard dev.)0.002782228 (±0.15731879)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 341.248 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3331.3331.333
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z341.248341.248341.248
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-4.0746.3010.003

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Supplemental data

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Sample components

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Entire RNAP-promoter initial transcribing complex with 5-nt RNA transcri...

EntireName: RNAP-promoter initial transcribing complex with 5-nt RNA transcript (RPitc-5nt)
Number of Components: 12

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Component #1: protein, RNAP-promoter initial transcribing complex with 5-nt RNA...

ProteinName: RNAP-promoter initial transcribing complex with 5-nt RNA transcript (RPitc-5nt)
Recombinant expression: No
SourceSpecies: Escherichia coli O157:H7 (bacteria)
Source (engineered)Expression System: Escherichia coli O157:H7 (bacteria)

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Component #2: protein, DNA-directed RNA polymerase subunit alpha

ProteinName: DNA-directed RNA polymerase subunit alphaPolymerase / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 36.55868 kDa
SourceSpecies: Escherichia coli O157:H7 (bacteria)
Source (engineered)Expression System: Escherichia coli O157:H7 (bacteria)

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Component #3: protein, DNA-directed RNA polymerase subunit beta

ProteinName: DNA-directed RNA polymerase subunit betaPolymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 150.820875 kDa
SourceSpecies: Escherichia coli O157:H7 (bacteria)
Source (engineered)Expression System: Escherichia coli O157:H7 (bacteria)

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Component #4: protein, DNA-directed RNA polymerase subunit beta'

ProteinName: DNA-directed RNA polymerase subunit beta'Polymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 155.366781 kDa
SourceSpecies: Escherichia coli O157:H7 (bacteria)
Source (engineered)Expression System: Escherichia coli O157:H7 (bacteria)

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Component #5: protein, DNA-directed RNA polymerase subunit omega

ProteinName: DNA-directed RNA polymerase subunit omegaPolymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 10.249547 kDa
SourceSpecies: Escherichia coli O157:H7 (bacteria)
Source (engineered)Expression System: Escherichia coli O157:H7 (bacteria)

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Component #6: protein, RNA polymerase sigma factor RpoD

ProteinName: RNA polymerase sigma factor RpoD / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 70.352242 kDa
SourceSpecies: Escherichia coli O157:H7 (bacteria)
Source (engineered)Expression System: Escherichia coli O157:H7 (bacteria)

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Component #7: nucleic-acid, synthetic non-template strand DNA (54-MER)

nucleic acidName: synthetic non-template strand DNA (54-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence: (DG)(DC)(DC)(DT)(DT)(DG)(DA)(DC)(DC)(DC) (DT)(DC)(DC)(DC)(DC)(DT)(DA)(DA)(DG)(DG) (DG)(DG)(DA)(DG)(DG)(DG)(DT)(DT)(DT)(DA) (DG)(DA)(DT)(DT)(DG)(DT)(DG)(DT)(DG)(DC) (DA)(DG)(DT)(DC)(DT)(DG) ...Sequence:
(DG)(DC)(DC)(DT)(DT)(DG)(DA)(DC)(DC)(DC) (DT)(DC)(DC)(DC)(DC)(DT)(DA)(DA)(DG)(DG) (DG)(DG)(DA)(DG)(DG)(DG)(DT)(DT)(DT)(DA) (DG)(DA)(DT)(DT)(DG)(DT)(DG)(DT)(DG)(DC) (DA)(DG)(DT)(DC)(DT)(DG)(DA)(DC)(DG)(DC) (DG)(DG)(DC)(DG)
MassTheoretical: 16.718658 kDa
SourceSpecies: Escherichia coli O157:H7 (bacteria)

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Component #8: nucleic-acid, synthetic template strand DNA (54-MER)

nucleic acidName: synthetic template strand DNA (54-MER) / Class: DNA / Structure: OTHER / Synthetic: No
Sequence: (DC)(DG)(DC)(DC)(DG)(DC)(DG)(DT)(DC)(DA) (DG)(DA)(DC)(DT)(DG)(DC)(DA)(DC)(DA)(DC) (DA)(DA)(DT)(DC)(DT)(DA)(DA)(DA)(DC)(DC) (DC)(DT)(DC)(DC)(DC)(DC)(DT)(DT)(DA)(DG) (DG)(DG)(DG)(DA)(DG)(DG) ...Sequence:
(DC)(DG)(DC)(DC)(DG)(DC)(DG)(DT)(DC)(DA) (DG)(DA)(DC)(DT)(DG)(DC)(DA)(DC)(DA)(DC) (DA)(DA)(DT)(DC)(DT)(DA)(DA)(DA)(DC)(DC) (DC)(DT)(DC)(DC)(DC)(DC)(DT)(DT)(DA)(DG) (DG)(DG)(DG)(DA)(DG)(DG)(DG)(DT)(DC)(DA) (DA)(DG)(DG)(DC)
MassTheoretical: 16.563607 kDa
SourceSpecies: Escherichia coli O157:H7 (bacteria)

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Component #9: nucleic-acid, RNA transcript (5-MER)

nucleic acidName: RNA transcript (5-MER) / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
(GTP)CAGU
MassTheoretical: 1.745967 kDa
SourceSpecies: Escherichia coli O157:H7 (bacteria)

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Component #10: ligand, CHAPSO

LigandName: CHAPSOCHAPS detergent / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 0.631884 kDa

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Component #11: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Component #12: ligand, ZINC ION

LigandName: ZINC ION / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 6.540905 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen State: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 5.8 mg/mL / pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen Name: ETHANE / Temperature: 295 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron Source: FIELD EMISSION GUN / Accelerating Voltage: 300 kV / Electron Dose: 50.46 e/Å2 / Illumination Mode: FLOOD BEAM
LensMagnification: 105000.0 X (nominal) / Cs: 2.7 mm / Imaging Mode: BRIGHT FIELD / Defocus: 1200.0 - 2200.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied Symmetry: C1 (asymmetric) / Number of Projections: 126197
3D reconstructionSoftware: cryoSPARC / Resolution: 2.7 Å / Resolution Method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Target Criteria: Correlation coefficient / Refinement space: REAL
Input PDB model: 6OUL
Output model

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