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- EMDB-5343: Molecular architecture of the human VP16-Mediator-RNA polymerase ... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-5343 | |||||||||
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Title | Molecular architecture of the human VP16-Mediator-RNA polymerase II-TFIIF assembly | |||||||||
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![]() | transcription / Mediator / RNA polymerase II / TFIIF / activator | |||||||||
Function / homology | ![]() RPB4-RPB7 complex / nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / RNA Polymerase I Transcription Initiation / 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 / RNA polymerase II transcribes snRNA genes / TP53 Regulates Transcription of DNA Repair Genes ...RPB4-RPB7 complex / nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / RNA Polymerase I Transcription Initiation / 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 / RNA polymerase II transcribes snRNA genes / TP53 Regulates Transcription of DNA Repair Genes / termination of RNA polymerase II transcription / 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 / RNA-templated transcription / RNA Polymerase II Pre-transcription Events / termination of RNA polymerase III transcription / Formation of TC-NER Pre-Incision Complex / termination of RNA polymerase I transcription / transcription initiation at RNA polymerase III promoter / RNA Polymerase I Promoter Escape / nucleolar large rRNA transcription by RNA polymerase I / maintenance of transcriptional fidelity during transcription elongation by RNA polymerase II / Gap-filling DNA repair synthesis and ligation in TC-NER / transcription initiation at RNA polymerase I promoter / transcription by RNA polymerase I / Estrogen-dependent gene expression / positive regulation of nuclear-transcribed mRNA poly(A) tail shortening / nuclear-transcribed mRNA catabolic process / transcription by RNA polymerase III / RNA polymerase II activity / Dual incision in TC-NER / transcription elongation by RNA polymerase I / tRNA transcription by RNA polymerase III / RNA polymerase I activity / RNA polymerase I complex / RNA polymerase III complex / positive regulation of translational initiation / transcription-coupled nucleotide-excision repair / RNA polymerase II, core complex / translesion synthesis / translation initiation factor binding / DNA-templated transcription initiation / transcription initiation at RNA polymerase II promoter / transcription elongation by RNA polymerase II / P-body / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / cytoplasmic stress granule / mRNA processing / ribosome biogenesis / single-stranded DNA binding / nucleic acid binding / transcription by RNA polymerase II / single-stranded RNA binding / protein dimerization activity / mRNA binding / nucleotide binding / nucleolus / mitochondrion / DNA binding / zinc ion binding / nucleoplasm / nucleus / metal ion binding / cytoplasm Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / negative staining / Resolution: 36.0 Å | |||||||||
![]() | Bernecky C / Grob P / Ebmeier CC / Nogales E / Taatjes DJ | |||||||||
![]() | ![]() 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 |
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Structure visualization
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 14.8 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 12.3 KB 12.3 KB | Display Display | ![]() |
Images | ![]() | 70.4 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 3j0kMC M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | none | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 4.29 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Assembly of VP16-bound human Mediator, RNA polymerase II, and TFIIF
Entire | Name: Assembly of VP16-bound human Mediator, RNA polymerase II, and TFIIF |
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Components |
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-Supramolecule #1000: Assembly of VP16-bound human Mediator, RNA polymerase II, and TFIIF
Supramolecule | Name: 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 |
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Molecular weight | Theoretical: 1.9 MDa |
-Macromolecule #1: core Mediator
Macromolecule | Name: 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 |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 1.2 MDa |
-Macromolecule #2: RNA polymerase II
Macromolecule | Name: 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 |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 520 KDa |
-Macromolecule #3: TFIIF
Macromolecule | Name: TFIIF / type: protein_or_peptide / ID: 3 / Name.synonym: TFIIF / Details: RAP74 and RAP30 expressed separately then combined / Oligomeric state: Dimer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 10 KDa |
Recombinant expression | Organism: ![]() ![]() |
-Experimental details
-Structure determination
Method | negative staining, cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.9 Details: 20 mM HEPES, 0.10 mM EDTA, 150 mM KCl, 0.02% NP-40, 35% glycerol |
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Staining | Type: 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) |
Grid | Details: thin carbon-coated holey carbon 400 mesh copper grid |
Vitrification | Cryogen 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
Microscope | FEI TECNAI F20 |
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Image recording | Category: 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 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 29000 |
Sample stage | Specimen holder: side entry / Specimen holder model: GATAN LIQUID NITROGEN |
Experimental equipment | ![]() Model: Tecnai F20 / Image courtesy: FEI Company |
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Image processing
Details | The particles were selected interactively at the computer terminal. |
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CTF correction | Details: each micrograph |
Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 36.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider / Number images used: 3146 |
-Atomic model buiding 1
Initial model | PDB ID: |
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Software | Name: Situs |
Details | Protocol: rigid body. the TFIIS chain S was removed before fitting |
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Target criteria: contour-based Laplacian correlation |
Output model | ![]() PDB-3j0k: |