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- EMDB-8732: Escherichia coli 6S RNA derivative in complex with Escherichia co... -

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Basic information

Entry
Database: EMDB / ID: 8732
TitleEscherichia coli 6S RNA derivative in complex with Escherichia coli RNA polymerase sigma70-holoenzyme
Map dataprimary map
SampleEscherichia coli 6S RNA derivative in complex with Escherichia coli RNA polymerase sigma70-holoenzyme
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • RNA polymerase sigma factor RpoD
  • nucleic-acidNucleic acid
  • (ligand) x 2
Function / homologyRNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor, region 2 / RPB6/omega subunit-like superfamily / RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase, beta subunit, external 1 domain / DNA-directed RNA polymerase, subunit 2 / RNA polymerase Rpb2, OB-fold / RNA polymerase sigma-70 like domain / RNA polymerase sigma factor, region 3/4-like / RNA polymerase, RBP11-like subunit ...RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor, region 2 / RPB6/omega subunit-like superfamily / RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase, beta subunit, external 1 domain / DNA-directed RNA polymerase, subunit 2 / RNA polymerase Rpb2, OB-fold / RNA polymerase sigma-70 like domain / RNA polymerase sigma factor, region 3/4-like / RNA polymerase, RBP11-like subunit / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase subunit, RPB6/omega / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / DNA-directed RNA polymerase, insert domain / RNA polymerase, alpha subunit, C-terminal / Winged helix-like DNA-binding domain superfamily / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb3/Rpb11 dimerisation domain / Sigma-70, non-essential region / Sigma-70, region 4 / Sigma-70 region 2 / Sigma-70 region 3 / Sigma-70 factor, region 1.1 / Bacterial RNA polymerase, alpha chain C terminal domain / RNA polymerase Rpb6 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb2, domain 6 / Sigma-70 factor, region 1.2 / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase sigma-70 region 1.2 / RNA polymerase Rpb2, domain 3 / RNA polymerase, subunit omega/K/RPB6 / RNA polymerases beta chain signature. / Sigma-70 factors family signature 2. / Sigma-70 factors family signature 1. / RNA polymerase beta subunit external 1 domain / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 3 / RNA polymerase beta subunit / RNA polymerase, alpha subunit / RNA polymerase sigma-70 / RNA polymerase, beta subunit, protrusion / DNA-directed RNA polymerase, omega subunit / RNA polymerase, N-terminal / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 7 / RNA polymerase sigma factor 70, non-essential domain / RNA polymerase sigma-70 region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 region 3 / RNA polymerase sigma factor 70, region 1.1 / RNA polymerase, beta subunit, conserved site / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb2, domain 7 / RNA polymerase complex / transcription initiation from bacterial-type RNA polymerase promoter / sigma factor activity / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein-containing complex assembly / response to heat / protein dimerization activity / transcription, DNA-templated / DNA-binding transcription factor activity / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm / RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta
Function and homology information
SourceEscherichia coli (E. coli) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / 3.78 Å resolution
AuthorsChen J / Darst SA
CitationJournal: Mol. Cell / Year: 2017
Title: 6S RNA Mimics B-Form DNA to Regulate Escherichia coli RNA Polymerase.
Authors: James Chen / Karen M Wassarman / Shili Feng / Katherine Leon / Andrey Feklistov / Jared T Winkelman / Zongli Li / Thomas Walz / Elizabeth A Campbell / Seth A Darst
Abstract: Noncoding RNAs (ncRNAs) regulate gene expression in all organisms. Bacterial 6S RNAs globally regulate transcription by binding RNA polymerase (RNAP) holoenzyme and competing with promoter DNA. ...Noncoding RNAs (ncRNAs) regulate gene expression in all organisms. Bacterial 6S RNAs globally regulate transcription by binding RNA polymerase (RNAP) holoenzyme and competing with promoter DNA. Escherichia coli (Eco) 6S RNA interacts specifically with the housekeeping σ-holoenzyme (Eσ) and plays a key role in the transcriptional reprogramming upon shifts between exponential and stationary phase. Inhibition is relieved upon 6S RNA-templated RNA synthesis. We report here the 3.8 Å resolution structure of a complex between 6S RNA and Eσ determined by single-particle cryo-electron microscopy and validation of the structure using footprinting and crosslinking approaches. Duplex RNA segments have A-form C3' endo sugar puckers but widened major groove widths, giving the RNA an overall architecture that mimics B-form promoter DNA. Our results help explain the specificity of Eco 6S RNA for Eσ and show how an ncRNA can mimic B-form DNA to directly regulate transcription by the DNA-dependent RNAP.
Validation ReportPDB-ID: 5vt0

SummaryFull reportAbout validation report
DateDeposition: May 15, 2017 / Header (metadata) release: Aug 9, 2017 / Map release: Nov 1, 2017 / Last update: Dec 6, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.017
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.017
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-5vt0
  • Surface level: 0.017
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_8732.map.gz (map file in CCP4 format, 67109 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
256 pix
1.3 Å/pix.
= 332.8 Å
256 pix
1.3 Å/pix.
= 332.8 Å
256 pix
1.3 Å/pix.
= 332.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.3 Å
Density
Contour Level:0.014 (by author), 0.017 (movie #1):
Minimum - Maximum-0.07076079 - 0.11960874
Average (Standard dev.)0.00019718395 (0.0041868477)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions256256256
Origin0.00.00.0
Limit255.0255.0255.0
Spacing256256256
CellA=B=C: 332.8 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.31.31.3
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z332.800332.800332.800
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0710.1200.000

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Supplemental data

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Sample components

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Entire Escherichia coli 6S RNA derivative in complex with Escherichia co...

EntireName: Escherichia coli 6S RNA derivative in complex with Escherichia coli RNA polymerase sigma70-holoenzyme
Number of components: 9

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Component #1: protein, Escherichia coli 6S RNA derivative in complex with Esche...

ProteinName: Escherichia coli 6S RNA derivative in complex with Escherichia coli RNA polymerase sigma70-holoenzyme
Recombinant expression: No
MassExperimental: 475 kDa
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (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: 26.330996 kDa
SourceSpecies: Escherichia coli (strain K12) (bacteria) / Strain: K12
Source (engineered)Expression System: Escherichia coli BL21(DE3) (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 (strain K12) (bacteria) / Strain: K12
Source (engineered)Expression System: Escherichia coli BL21(DE3) (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 (strain K12) (bacteria) / Strain: K12
Source (engineered)Expression System: Escherichia coli BL21(DE3) (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 (strain K12) (bacteria) / Strain: K12
Source (engineered)Expression System: Escherichia coli BL21(DE3) (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: 60.464371 kDa
SourceSpecies: Escherichia coli (strain K12) (bacteria) / Strain: K12
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #7: nucleic-acid, Escherichia coli 6S RNA derivative (112-MER)

Nucleic-acidName: Escherichia coli 6S RNA derivative (112-MER) / Class: RNA / Details: prepared by in vitro transcription / Structure: OTHER / Synthetic: No
Sequence:
GGACUCCCAG UCGGCACAUG CGAUAUUUCA UACCACAAGA AUGUGGCGCU CCGCGGUUGG UGAGCAUGCU CGGUCCGUCC GAGAAGCCUU AAAACUGCGA CGACACAUUC ACCUUGAACC AAGGCGUGUA CCGUUACAGG GGUC
MassTheoretical: 46.3085 kDa
SourceSpecies: Escherichia coli (E. coli)

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Component #8: ligand, MAGNESIUM ION

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

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Component #9: 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 solutionpH: 8
VitrificationCryogen name: ETHANE

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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: 1.8 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 362926
3D reconstructionResolution: 3.78 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Target criteria: Correlation coefficient / Refinement space: REAL
Output model

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