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- EMDB-28110: Cryo-EM structure of consensus elemental paused elongation comple... -

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Basic information

Entry
Database: EMDB / ID: EMD-28110
TitleCryo-EM structure of consensus elemental paused elongation complex with a folded TL
Map dataa postprocessed map
Sample
  • Complex: consensus elemental paused elongation complex with a folded TL
    • DNA: non-template DNA
    • DNA: template DNA
    • RNA: RNA
    • Protein or peptide: DNA-directed RNA polymerase subunit alphaPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit betaPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit beta'Polymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit omegaPolymerase
  • Ligand: CHAPSOCHAPS detergent
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
Function / homology
Function and homology information


DNA-directed RNA polymerase complex / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm
Similarity search - Function
DNA-directed RNA polymerase, omega subunit / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / RNA polymerase, alpha subunit, C-terminal / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase beta subunit, bacterial-type / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase beta subunit external 1 domain / RNA polymerase Rpb1, domain 3 superfamily ...DNA-directed RNA polymerase, omega subunit / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / RNA polymerase, alpha subunit, C-terminal / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase beta subunit, bacterial-type / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase beta subunit external 1 domain / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb6 / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb2, domain 2 superfamily / RPB6/omega subunit-like superfamily / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 4 / RNA polymerase, N-terminal / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb1, domain 5 / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / RNA polymerase Rpb2, domain 2 / RNA polymerase, beta subunit, protrusion / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase beta subunit / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, OB-fold / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerases beta chain signature. / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / DNA-directed RNA polymerase, subunit 2 / RNA polymerase Rpb2, domain 6 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily
Similarity search - Domain/homology
DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsKang JY / Chen J / Llewellyn E / Landick R / Darst SA
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118130 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM38330 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: An ensemble of interconverting conformations of the elemental paused transcription complex creates regulatory options.
Authors: Jin Young Kang / Tatiana V Mishanina / Yu Bao / James Chen / Eliza Llewellyn / James Liu / Seth A Darst / Robert Landick /
Abstract: Transcriptional pausing underpins the regulation of cellular RNA synthesis, but its mechanism remains incompletely understood. Sequence-specific interactions of DNA and RNA with the dynamic, ...Transcriptional pausing underpins the regulation of cellular RNA synthesis, but its mechanism remains incompletely understood. Sequence-specific interactions of DNA and RNA with the dynamic, multidomain RNA polymerase (RNAP) trigger reversible conformational changes at pause sites that temporarily interrupt the nucleotide addition cycle. These interactions initially rearrange the elongation complex (EC) into an elemental paused EC (ePEC). ePECs can form longer-lived PECs by further rearrangements or interactions of diffusible regulators. For both bacterial and mammalian RNAPs, a half-translocated state in which the next DNA template base fails to load into the active site appears central to the ePEC. Some RNAPs also swivel interconnected modules that may stabilize the ePEC. However, it is unclear whether swiveling and half-translocation are requisite features of a single ePEC state or if multiple ePEC states exist. Here, we use cryo-electron microscopy (cryo-EM) analysis of ePECs with different RNA-DNA sequences combined with biochemical probes of ePEC structure to define an interconverting ensemble of ePEC states. ePECs occupy either pre- or half-translocated states but do not always swivel, indicating that difficulty in forming the posttranslocated state at certain RNA-DNA sequences may be the essence of the ePEC. The existence of multiple ePEC conformations has broad implications for transcriptional regulation.
History
DepositionSep 12, 2022-
Header (metadata) releaseMar 1, 2023-
Map releaseMar 1, 2023-
UpdateMar 1, 2023-
Current statusMar 1, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28110.png

Downloads & links

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Map

FileDownload / File: emd_28110.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationa postprocessed map
Projections & slices

Image control

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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.027
Minimum - Maximum-0.11206071 - 0.18396206
Average (Standard dev.)-5.0438553e-05 (±0.0065832622)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 332.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_28110_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: first half map

Fileemd_28110_half_map_1.map
Annotationfirst half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: second half map

Fileemd_28110_half_map_2.map
Annotationsecond half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : consensus elemental paused elongation complex with a folded TL

EntireName: consensus elemental paused elongation complex with a folded TL
Components
  • Complex: consensus elemental paused elongation complex with a folded TL
    • DNA: non-template DNA
    • DNA: template DNA
    • RNA: RNA
    • Protein or peptide: DNA-directed RNA polymerase subunit alphaPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit betaPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit beta'Polymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit omegaPolymerase
  • Ligand: CHAPSOCHAPS detergent
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION

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Supramolecule #1: consensus elemental paused elongation complex with a folded TL

SupramoleculeName: consensus elemental paused elongation complex with a folded TL
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#7
Details: An RNA polymerase elongation complex was assembled at the consensus elemental pausing site. The complexes exhibit two conformations, and this is one of them having a folded trigger loop (TL).
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 450 KDa

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Macromolecule #1: non-template DNA

MacromoleculeName: non-template DNA / type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 9.793289 KDa
SequenceString:

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Macromolecule #2: template DNA

MacromoleculeName: template DNA / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 9.842328 KDa
SequenceString:

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Macromolecule #3: RNA

MacromoleculeName: RNA / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 5.360133 KDa
SequenceString:

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Macromolecule #4: DNA-directed RNA polymerase subunit alpha

MacromoleculeName: DNA-directed RNA polymerase subunit alpha / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 26.459125 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:

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Macromolecule #5: DNA-directed RNA polymerase subunit beta

MacromoleculeName: DNA-directed RNA polymerase subunit beta / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 150.820875 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:

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Macromolecule #6: DNA-directed RNA polymerase subunit beta'

MacromoleculeName: DNA-directed RNA polymerase subunit beta' / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 155.237672 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:

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Macromolecule #7: DNA-directed RNA polymerase subunit omega

MacromoleculeName: DNA-directed RNA polymerase subunit omega / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 10.249547 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:

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Macromolecule #8: CHAPSO

MacromoleculeName: CHAPSO / type: ligand / ID: 8 / Number of copies: 2 / Formula: 1N7
Molecular weightTheoretical: 631.884 Da
Chemical component information

ChemComp-1N7:
CHAPSO / detergent*YM / CHAPS detergent

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Macromolecule #9: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #10: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 10 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration8 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
20.0 mMTris
150.0 mMpotassium glutamate
5.0 mMmagnesium chlorideMgCl2
5.0 mMdithiothreitol
GridModel: C-flat-1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 22 K / Instrument: FEI VITROBOT MARK IV / Details: blot force 0, blot time 3 s.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Number real images: 2525 / Average exposure time: 10.0 sec. / Average electron dose: 47.34 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 716722
Startup modelType of model: OTHER
Details: Ab initio map was reconstructed in Cryosparc2 using the picked particles.
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 2)
Final 3D classificationNumber classes: 6 / Avg.num./class: 67850 / Software - Name: cryoSPARC
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 197891

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Atomic model buiding 1

Initial model
PDB IDChain

6c6t

chain_id: G, residue_range: 7-234

6c6t

chain_id: H, residue_range: 4-232

6c6t

chain_id: I, residue_range: 2-1342

6c6t

chain_id: J, residue_range: 16-1373

6c6t

chain_id: K, residue_range: 2-84
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8eg8:
Cryo-EM structure of consensus elemental paused elongation complex with a folded TL

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