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- EMDB-5344: Human Mediator-RNA polymerase II-TFIIF assembly in the absence of... -

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Basic information

Entry
Database: EMDB / ID: EMD-5344
TitleHuman Mediator-RNA polymerase II-TFIIF assembly in the absence of activator
Map dataactivator-free Mediator bound to pol II & TFIIF
Sample
  • Sample: Assembly of human Mediator, RNA polymerase II, and TFIIF
  • Protein or peptide: core Mediator
  • Protein or peptide: RNA polymerase II
  • Protein or peptide: TFIIFTranscription factor II F
Keywordstranscription / Mediator / RNA polymerase II / TFIIF / activator
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 32.0 Å
AuthorsBernecky C / Taatjes DJ
CitationJournal: J Mol Biol / Year: 2012
Title: Activator-mediator binding stabilizes RNA polymerase II orientation within the human mediator-RNA polymerase II-TFIIF assembly.
Authors: Carrie Bernecky / Dylan J Taatjes /
Abstract: The human Mediator complex controls RNA polymerase II (pol II) function in ways that remain incompletely understood. Activator-Mediator binding alters Mediator structure, and these activator-induced ...The human Mediator complex controls RNA polymerase II (pol II) function in ways that remain incompletely understood. Activator-Mediator binding alters Mediator structure, and these activator-induced structural shifts appear to play key roles in regulating transcription. A recent cryo-electron microscopy (EM) analysis revealed that pol II adopted a stable orientation within a Mediator-pol II-TFIIF assembly in which Mediator was bound to the activation domain of viral protein 16 (VP16). Whereas TFIIF was shown to be important for orienting pol II within this assembly, the potential role of the activator was not assessed. To determine how activator binding might affect pol II orientation, we isolated human Mediator-pol II-TFIIF complexes in which Mediator was not bound to an activator. Cryo-EM analysis of this assembly, coupled with pol II crystal structure docking, revealed that pol II binds Mediator at the same general location; however, in contrast to VP16-bound Mediator, pol II does not appear to stably orient in the absence of an activator. Variability in pol II orientation might be important mechanistically, perhaps to enable sense and antisense transcription at human promoters. Because Mediator interacts extensively with pol II, these results suggest that Mediator structural shifts induced by activator binding help stably orient pol II prior to transcription initiation.
History
DepositionOct 4, 2011-
Header (metadata) releaseNov 28, 2011-
Map releaseFeb 23, 2012-
UpdateFeb 23, 2012-
Current statusFeb 23, 2012Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0157
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0157
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5344_1.png

Downloads & links

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Map

FileDownload / File: emd_5344.map.gz / Format: CCP4 / Size: 15.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationactivator-free Mediator bound to pol II & TFIIF
Voxel sizeX=Y=Z: 4.22 Å
Density
Contour LevelBy AUTHOR: 0.0157397 / Movie #1: 0.0157
Minimum - Maximum-0.00709368 - 0.06922273
Average (Standard dev.)0.00026938 (±0.00288081)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions161161161
Spacing161161161
CellA=B=C: 679.42 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.224.224.22
M x/y/z161161161
origin x/y/z0.0000.0000.000
length x/y/z679.420679.420679.420
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS161161161
D min/max/mean-0.0070.0690.000

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Supplemental data

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Sample components

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Entire : Assembly of human Mediator, RNA polymerase II, and TFIIF

EntireName: Assembly of human Mediator, RNA polymerase II, and TFIIF
Components
  • Sample: Assembly of human Mediator, RNA polymerase II, and TFIIF
  • Protein or peptide: core Mediator
  • Protein or peptide: RNA polymerase II
  • Protein or peptide: TFIIFTranscription factor II F

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Supramolecule #1000: Assembly of human Mediator, RNA polymerase II, and TFIIF

SupramoleculeName: Assembly of human Mediator, RNA polymerase II, and TFIIF
type: sample / ID: 1000
Oligomeric state: one Mediator complex binds one pol II-TFIIF
Number unique components: 3
Molecular weightTheoretical: 1.9 MDa

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Macromolecule #1: core Mediator

MacromoleculeName: core Mediator / type: protein_or_peptide / ID: 1 / Name.synonym: Mediator
Details: affinity purified using an antibody to the MED26 subunit
Oligomeric state: 26 subunit complex / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Homo sapiens (human) / synonym: human / Cell: HeLa / Organelle: Nucleus
Molecular weightTheoretical: 1.2 MDa

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Macromolecule #2: RNA polymerase II

MacromoleculeName: RNA polymerase II / type: protein_or_peptide / ID: 2 / Name.synonym: pol II / Details: unphosphorylated / Oligomeric state: 12-subunit complex / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: HeLa / Organelle: Nucleus
Molecular weightTheoretical: 520 KDa

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Macromolecule #3: TFIIF

MacromoleculeName: TFIIF / type: protein_or_peptide / ID: 3 / Name.synonym: TFIIF / Details: RAP74 and RAP30 expressed separately then combined / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 10 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.9
Details: 20 mM HEPES, 0.10 mM EDTA, 150 mM KCl, 0.02% NP-40, 35% glycerol
StainingType: NEGATIVE
Details: grids with adsorbed protein washed 3x with buffer containing 5% trehalose, 20 mM HEPES, 100 mM KCl, and 0.10 mM EDTA, then subject to cryo-negative staining in a saturated solution (1.2M) of ...Details: grids with adsorbed protein washed 3x with buffer containing 5% trehalose, 20 mM HEPES, 100 mM KCl, and 0.10 mM EDTA, then subject to cryo-negative staining in a saturated solution (1.2M) of ammonium molybdate (pH 7.5)
GridDetails: thin carbon-coated holey carbon 400 mesh copper grid
VitrificationCryogen name: ETHANE / Chamber temperature: 90 K / Instrument: OTHER
Method: blot for 2 seconds, dry for 3 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 29000
Sample stageSpecimen holder: side entry / Specimen holder model: GATAN LIQUID NITROGEN
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 12.7 µm / Number real images: 139 / Average electron dose: 15 e/Å2 / Od range: 1 / Bits/pixel: 16
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 32.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider / Number images used: 5489
DetailsThe particles were selected interactively at the computer terminal.

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Atomic model buiding 1

Initial modelPDB ID:

1y1v

SoftwareName: Situs
DetailsProtocol: rigid body. the TFIIS chain S was removed before fitting
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: contour-based Laplacian correlation

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