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- EMDB-5343: Molecular architecture of the human VP16-Mediator-RNA polymerase ... -

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Basic information

Entry
Database: EMDB / ID: EMD-5343
TitleMolecular architecture of the human VP16-Mediator-RNA polymerase II-TFIIF assembly
Map datanone
Sample
  • Sample: Assembly of VP16-bound 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
Function / homology
Function and homology information


RPB4-RPB7 complex / : / nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / : / RNA Polymerase I Transcription Initiation / positive regulation of nuclear-transcribed mRNA poly(A) tail shortening / Processing of Capped Intron-Containing Pre-mRNA / : / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA Pol II CTD phosphorylation and interaction with CE ...RPB4-RPB7 complex / : / nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / : / RNA Polymerase I Transcription Initiation / positive regulation of nuclear-transcribed mRNA poly(A) tail shortening / Processing of Capped Intron-Containing Pre-mRNA / : / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA Pol II CTD phosphorylation and interaction with CE / Formation of the Early Elongation Complex / mRNA Capping / termination of RNA polymerase II transcription / Formation of TC-NER Pre-Incision Complex / RNA polymerase II transcribes snRNA genes / RNA Polymerase I Promoter Escape / TP53 Regulates Transcription of DNA Repair Genes / Estrogen-dependent gene expression / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / RNA Polymerase II Transcription Initiation / RNA Polymerase II Transcription Initiation And Promoter Clearance / maintenance of transcriptional fidelity during transcription elongation by RNA polymerase II / RNA-templated transcription / termination of RNA polymerase III transcription / RNA Polymerase II Pre-transcription Events / Dual incision in TC-NER / RNA polymerase I activity / termination of RNA polymerase I transcription / transcription initiation at RNA polymerase III promoter / tRNA transcription by RNA polymerase III / nucleolar large rRNA transcription by RNA polymerase I / Gap-filling DNA repair synthesis and ligation in TC-NER / transcription initiation at RNA polymerase I promoter / RNA polymerase II activity / transcription elongation by RNA polymerase I / positive regulation of translational initiation / transcription-coupled nucleotide-excision repair / translesion synthesis / RNA polymerase I complex / RNA polymerase III complex / transcription by RNA polymerase I / transcription by RNA polymerase III / RNA polymerase II, core complex / translation initiation factor binding / transcription elongation by RNA polymerase II / P-body / DNA-templated transcription initiation / transcription initiation at RNA polymerase II promoter / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / cytoplasmic stress granule / single-stranded DNA binding / ribosome biogenesis / transcription by RNA polymerase II / single-stranded RNA binding / nucleic acid binding / protein dimerization activity / nucleotide binding / mRNA binding / nucleolus / mitochondrion / DNA binding / zinc ion binding / nucleoplasm / metal ion binding / nucleus / cytoplasm
Similarity search - Function
DNA-directed RNA polymerase II subunit Rpb4-like / RNA polymerase Rpb4/RPC9, core / DNA-directed RNA-polymerase II subunit / RNA polymerase II, heptapeptide repeat, eukaryotic / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1 C-terminal repeat / Eukaryotic RNA polymerase II heptapeptide repeat. / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 7 superfamily ...DNA-directed RNA polymerase II subunit Rpb4-like / RNA polymerase Rpb4/RPC9, core / DNA-directed RNA-polymerase II subunit / RNA polymerase II, heptapeptide repeat, eukaryotic / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1 C-terminal repeat / Eukaryotic RNA polymerase II heptapeptide repeat. / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 7 superfamily / RNA polymerase Rpb1, domain 7 / Pol II subunit B9, C-terminal zinc ribbon / RNA polymerase RBP11 / Rpb4/RPC9 superfamily / RNA polymerase subunit Rpb4/RPC9 / RNA polymerase Rpb4 / Zinc finger TFIIS-type signature. / HRDC-like superfamily / RNA polymerase subunit Rpb7-like / RNA polymerase Rpb7-like , N-terminal / RNA polymerase Rpb7-like, N-terminal domain superfamily / SHS2 domain found in N terminus of Rpb7p/Rpc25p/MJ0397 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 5 / DNA-directed RNA polymerase subunit RPABC5/Rpb10 / RNA polymerases, subunit N, zinc binding site / RNA polymerase subunit RPB10 / RNA polymerases N / 8 kDa subunit / RNA polymerases N / 8 Kd subunits signature. / DNA-directed RNA polymerase, M/15kDa subunit / RNA polymerases M/15 Kd subunit / RNA polymerase subunit 9 / DNA-directed RNA polymerase M, 15kDa subunit, conserved site / RNA polymerases M / 15 Kd subunits signature. / DNA-directed RNA polymerase subunit/transcription factor S / RNA polymerase, Rpb8 / RNA polymerase Rpb8 / RNA polymerase subunit 8 / RNA polymerase, Rpb5, N-terminal / RNA polymerase Rpb5, N-terminal domain superfamily / RNA polymerase Rpb5, N-terminal domain / DNA-directed RNA polymerase, subunit RPB6 / DNA-directed RNA polymerase subunit Rpo11 / RNA polymerase, subunit H/Rpb5, conserved site / RNA polymerases H / 23 Kd subunits signature. / DNA-directed RNA polymerase Rpb11, 13-16kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo5/Rpb5 / RNA polymerases L / 13 to 16 Kd subunits signature. / DNA-directed RNA polymerase, 30-40kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo3/Rpb3/RPAC1 / RNA polymerases D / 30 to 40 Kd subunits signature. / DNA-directed RNA polymerase, RBP11-like dimerisation domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase, subunit H/Rpb5 C-terminal / RPB5-like RNA polymerase subunit superfamily / RNA polymerase Rpb5, C-terminal domain / Archaeal Rpo6/eukaryotic RPB6 RNA polymerase subunit / DNA-directed RNA polymerase, 14-18kDa subunit, conserved site / RNA polymerases K / 14 to 18 Kd subunits signature. / Zinc finger, TFIIS-type / Transcription factor S-II (TFIIS) / Zinc finger TFIIS-type profile. / C2C2 Zinc finger / Ribosomal protein S1-like RNA-binding domain / S1 RNA binding domain / S1 domain / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb1, clamp domain superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 4 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 5 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2
Similarity search - Domain/homology
DNA-directed RNA polymerase II subunit RPB1 / DNA-directed RNA polymerase II subunit RPB2 / DNA-directed RNA polymerase II subunit RPB3 / DNA-directed RNA polymerase II subunit RPB4 / DNA-directed RNA polymerases I, II, and III subunit RPABC1 / DNA-directed RNA polymerases I, II, and III subunit RPABC2 / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / DNA-directed RNA polymerases I, II, and III subunit RPABC5 / DNA-directed RNA polymerase II subunit RPB9 / DNA-directed RNA polymerase II subunit RPB7 / DNA-directed RNA polymerase II subunit RPB11
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 36.0 Å
AuthorsBernecky C / Grob P / Ebmeier CC / Nogales E / Taatjes DJ
CitationJournal: PLoS Biol / Year: 2011
Title: Molecular architecture of the human Mediator-RNA polymerase II-TFIIF assembly.
Authors: Carrie Bernecky / Patricia Grob / Christopher C Ebmeier / Eva Nogales / Dylan J Taatjes /
Abstract: The macromolecular assembly required to initiate transcription of protein-coding genes, known as the Pre-Initiation Complex (PIC), consists of multiple protein complexes and is approximately 3.5 MDa ...The macromolecular assembly required to initiate transcription of protein-coding genes, known as the Pre-Initiation Complex (PIC), consists of multiple protein complexes and is approximately 3.5 MDa in size. At the heart of this assembly is the Mediator complex, which helps regulate PIC activity and interacts with the RNA polymerase II (pol II) enzyme. The structure of the human Mediator-pol II interface is not well-characterized, whereas attempts to structurally define the Mediator-pol II interaction in yeast have relied on incomplete assemblies of Mediator and/or pol II and have yielded inconsistent interpretations. We have assembled the complete, 1.9 MDa human Mediator-pol II-TFIIF complex from purified components and have characterized its structural organization using cryo-electron microscopy and single-particle reconstruction techniques. The orientation of pol II within this assembly was determined by crystal structure docking and further validated with projection matching experiments, allowing the structural organization of the entire human PIC to be envisioned. Significantly, pol II orientation within the Mediator-pol II-TFIIF assembly can be reconciled with past studies that determined the location of other PIC components relative to pol II itself. Pol II surfaces required for interacting with TFIIB, TFIIE, and promoter DNA (i.e., the pol II cleft) are exposed within the Mediator-pol II-TFIIF structure; RNA exit is unhindered along the RPB4/7 subunits; upstream and downstream DNA is accessible for binding additional factors; and no major structural re-organization is necessary to accommodate the large, multi-subunit TFIIH or TFIID complexes. The data also reveal how pol II binding excludes Mediator-CDK8 subcomplex interactions and provide a structural basis for Mediator-dependent control of PIC assembly and function. Finally, parallel structural analysis of Mediator-pol II complexes lacking TFIIF reveal that TFIIF plays a key role in stabilizing pol II orientation within the assembly.
History
DepositionOct 4, 2011-
Header (metadata) releaseOct 13, 2011-
Map releaseOct 13, 2011-
UpdateOct 17, 2011-
Current statusOct 17, 2011Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0284
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0284
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j0k
  • Surface level: 0.0284
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3j0k
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5343_1.png

Downloads & links

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Map

FileDownload / File: emd_5343.map.gz / Format: CCP4 / Size: 15.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationnone
Voxel sizeX=Y=Z: 4.29 Å
Density
Contour LevelBy AUTHOR: 0.0284 / Movie #1: 0.0284
Minimum - Maximum-0.00885565 - 0.0734321
Average (Standard dev.)0.000528866 (±0.00409114)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions161161161
Spacing161161161
CellA=B=C: 690.69 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.294.294.29
M x/y/z161161161
origin x/y/z0.0000.0000.000
length x/y/z690.690690.690690.690
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-0.0090.0730.001

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Supplemental data

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Sample components

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Entire : Assembly of VP16-bound human Mediator, RNA polymerase II, and TFIIF

EntireName: Assembly of VP16-bound human Mediator, RNA polymerase II, and TFIIF
Components
  • Sample: Assembly of VP16-bound 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 VP16-bound human Mediator, RNA polymerase II, and TFIIF

SupramoleculeName: Assembly of VP16-bound 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: bound to GST-VP16 (residues 411 - 490) / 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 subjected to cryo-negative staining in a saturated solution (1.2M) ...Details: grids with adsorbed protein washed 3x with buffer containing 5% trehalose, 20 mM HEPES, 100 mM KCl, and 0.10 mM EDTA, then subjected 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: 4.5 µm / Nominal defocus min: 1.0 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: OTHER / Digitization - Sampling interval: 12.9 µm / Number real images: 106 / 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: 36.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider / Number images used: 3146
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
Output model

PDB-3j0k:
Orientation of RNA polymerase II within the human VP16-Mediator-pol II-TFIIF assembly

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