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- EMDB-2367: Structural mimicry in transcription regulation of human RNA polym... -

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Basic information

Entry
Database: EMDB / ID: EMD-2367
TitleStructural mimicry in transcription regulation of human RNA polymerase II by the DNA helicase RECQL5
Map dataReconstruction of elongating human Pol II in complex with the DNA helicase RECQL5
Sample
  • Sample: Human Pol II in complex with artificial transcription bubble and RECQL5DNA polymerase II
  • Protein or peptide: RNA polymerase II
  • Protein or peptide: RECQL5
  • RNA: transcription bubble RNA strand
  • DNA: non-template strandCoding strand
  • DNA: template strandTranscription (biology)
Keywordstranscription regulation / RNA polymerase II / RecQ helicase / RECQL5 / DNA repair
Biological speciesHomo sapiens (human) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 13.0 Å
AuthorsKassube SA / Jinek M / Fang J / Tsutakawa S / Nogales E
CitationJournal: Nat Struct Mol Biol / Year: 2013
Title: Structural mimicry in transcription regulation of human RNA polymerase II by the DNA helicase RECQL5.
Authors: Susanne A Kassube / Martin Jinek / Jie Fang / Susan Tsutakawa / Eva Nogales /
Abstract: RECQL5 is a member of the highly conserved RecQ family of DNA helicases involved in DNA repair. RECQL5 interacts with RNA polymerase II (Pol II) and inhibits transcription of protein-encoding genes ...RECQL5 is a member of the highly conserved RecQ family of DNA helicases involved in DNA repair. RECQL5 interacts with RNA polymerase II (Pol II) and inhibits transcription of protein-encoding genes by an unknown mechanism. We show that RECQL5 contacts the Rpb1 jaw domain of Pol II at a site that overlaps with the binding site for the transcription elongation factor TFIIS. Our cryo-EM structure of elongating Pol II arrested in complex with RECQL5 shows that the RECQL5 helicase domain is positioned to sterically block elongation. The crystal structure of the RECQL5 KIX domain reveals similarities with TFIIS, and binding of RECQL5 to Pol II interferes with the ability of TFIIS to promote transcriptional read-through in vitro. Together, our findings reveal a dual mode of transcriptional repression by RECQL5 that includes structural mimicry of the Pol II-TFIIS interaction.
History
DepositionApr 22, 2013-
Header (metadata) releaseMay 29, 2013-
Map releaseJun 19, 2013-
UpdateJul 17, 2013-
Current statusJul 17, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.00547
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.00547
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2367-REC...

Downloads & links

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Map

FileDownload / File: emd_2367.map.gz / Format: CCP4 / Size: 20.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of elongating human Pol II in complex with the DNA helicase RECQL5
Voxel sizeX=Y=Z: 2.31 Å
Density
Contour LevelBy AUTHOR: 0.00547 / Movie #1: 0.00547
Minimum - Maximum-0.03588435 - 0.0539254
Average (Standard dev.)0.00009505 (±0.00289447)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions176176176
Spacing176176176
CellA=B=C: 406.56 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.312.312.31
M x/y/z176176176
origin x/y/z0.0000.0000.000
length x/y/z406.560406.560406.560
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS176176176
D min/max/mean-0.0360.0540.000

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Supplemental data

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Sample components

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Entire : Human Pol II in complex with artificial transcription bubble and ...

EntireName: Human Pol II in complex with artificial transcription bubble and RECQL5DNA polymerase II
Components
  • Sample: Human Pol II in complex with artificial transcription bubble and RECQL5DNA polymerase II
  • Protein or peptide: RNA polymerase II
  • Protein or peptide: RECQL5
  • RNA: transcription bubble RNA strand
  • DNA: non-template strandCoding strand
  • DNA: template strandTranscription (biology)

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Supramolecule #1000: Human Pol II in complex with artificial transcription bubble and ...

SupramoleculeName: Human Pol II in complex with artificial transcription bubble and RECQL5
type: sample / ID: 1000
Oligomeric state: 12-subunit Pol II complex bound to transcription bubble and monomeric RECQL5
Number unique components: 5
Molecular weightTheoretical: 615 KDa

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Macromolecule #1: RNA polymerase II

MacromoleculeName: RNA polymerase II / type: protein_or_peptide / ID: 1 / Name.synonym: Pol II / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / Strain: HeLa / synonym: Human / Organelle: Nucleus
Molecular weightTheoretical: 517 KDa

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Macromolecule #5: RECQL5

MacromoleculeName: RECQL5 / type: protein_or_peptide / ID: 5 / Name.synonym: RecQ5 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 69.3 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pGEX-6P-1

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Macromolecule #2: transcription bubble RNA strand

MacromoleculeName: transcription bubble RNA strand / type: rna / ID: 2 / Classification: OTHER / Structure: OTHER / Synthetic?: No
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 6.1 KDa
SequenceString:
UAUAUGCAUA AAGACCAGGC

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Macromolecule #3: non-template strand

MacromoleculeName: non-template strand / type: dna / ID: 3 / Classification: DNA / Structure: OTHER / Synthetic?: No
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 13.3 KDa
SequenceString:
TAGTAAACTA GTATTGAAAG TACTTGAGCT TAGACAGCAT GTC

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Macromolecule #4: template strand

MacromoleculeName: template strand / type: dna / ID: 4 / Classification: DNA / Structure: OTHER / Synthetic?: No
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 8.5 KDa
SequenceString:
CTCAAGTACT TACGCCTGGT CATTACTA

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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

Concentration0.037 mg/mL
BufferpH: 8
Details: 20 mM Hepes pH 8.0, 4mM MgCl2, 50 mM KCl, 0.05% NP-40, 1mM TCEP
GridDetails: 400-mesh C-flats (Protochips Inc.) with thin carbon support (Protochips Inc.)
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 86 K / Instrument: FEI VITROBOT MARK II / Method: blot for 2 sec, 0 offset

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 2.8 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 100000
Sample stageSpecimen holder: Side-entry cryostage / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 78 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
Legacy - Electron beam tilt params: 0
DateOct 6, 2012
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Number real images: 5224 / Average electron dose: 20 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: whole micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2, SPARX / Number images used: 121416
DetailsImage processing was performed in the Appion processing environment. 3D reconstruction was performed using EMAN2 and SPARX libraries. Final map was filtered to local resolution using the blocres function of the Bsoft package.

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Atomic model buiding 1

Initial modelPDB ID:

1y1w

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

2wwy

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 3

Initial modelPDB ID:

4bk0

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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