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- EMDB-5407: Cryo-EM map of a yeast minimal preinitiation complex interacting ... -

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Entry
Database: EMDB / ID: EMD-5407
TitleCryo-EM map of a yeast minimal preinitiation complex interacting with the Mediator Head module
Map dataCryo-EM map of a complex including RNA polymerase II, Mediator Head module, TFIIF, TFIIB, TBP, and promoter DNA. TFIIB, TBP, and DNA are not visible in the map and are presumed to be disordered
Sample
  • Sample: Complex of RNA polymerase II, Mediator Head module, TFIIF, TFIIB, TBP, and promoter DNA
  • Protein or peptide: RNA polymerase II
  • Protein or peptide: Head module
  • Protein or peptide: TFIIF
Keywordstranscription / RNA polymerase II / Mediator / Head module / preinitiation complex
Function / homology
Function and homology information


RNA polymerase II complex recruiting activity / transcription open complex formation at RNA polymerase II promoter / TFIIH-class transcription factor complex binding / core mediator complex / negative regulation of ribosomal protein gene transcription by RNA polymerase II / RPB4-RPB7 complex / mediator complex / nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / RNA Polymerase I Transcription Initiation / Processing of Capped Intron-Containing Pre-mRNA ...RNA polymerase II complex recruiting activity / transcription open complex formation at RNA polymerase II promoter / TFIIH-class transcription factor complex binding / core mediator complex / negative regulation of ribosomal protein gene transcription by RNA polymerase II / RPB4-RPB7 complex / mediator 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 / termination of RNA polymerase II transcription / TP53 Regulates Transcription of DNA Repair Genes / 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 / termination of RNA polymerase III transcription / RNA Polymerase II Pre-transcription Events / RNA-templated transcription / positive regulation of nuclear-transcribed mRNA poly(A) tail shortening / Formation of TC-NER Pre-Incision Complex / transcription initiation at RNA polymerase III promoter / RNA Polymerase I Promoter Escape / termination of RNA polymerase I transcription / transcription initiation at RNA polymerase I promoter / nucleolar large rRNA transcription by RNA polymerase I / Gap-filling DNA repair synthesis and ligation in TC-NER / TFIID-class transcription factor complex binding / Estrogen-dependent gene expression / maintenance of transcriptional fidelity during transcription elongation by RNA polymerase II / nuclear-transcribed mRNA catabolic process / positive regulation of translational initiation / transcription by RNA polymerase III / Dual incision in TC-NER / positive regulation of transcription initiation by RNA polymerase II / translesion synthesis / RNA polymerase I complex / transcription elongation by RNA polymerase I / RNA polymerase III complex / cellular response to nutrient levels / RNA polymerase II core promoter sequence-specific DNA binding / RNA polymerase II preinitiation complex assembly / RNA polymerase II, core complex / tRNA transcription by RNA polymerase III / transcription by RNA polymerase I / translation initiation factor binding / transcription-coupled nucleotide-excision repair / TBP-class protein binding / transcription coregulator activity / transcription initiation at RNA polymerase II promoter / transcription elongation by RNA polymerase II / P-body / DNA-templated transcription initiation / positive regulation of transcription elongation by RNA polymerase II / ribonucleoside binding / mRNA processing / DNA-directed RNA polymerase / cytoplasmic stress granule / DNA-directed RNA polymerase activity / transcription corepressor activity / peroxisome / single-stranded DNA binding / ribosome biogenesis / cellular response to heat / protein-macromolecule adaptor activity / DNA-binding transcription factor binding / nucleic acid binding / transcription by RNA polymerase II / transcription coactivator activity / single-stranded RNA binding / protein dimerization activity / RNA polymerase II cis-regulatory region sequence-specific DNA binding / protein domain specific binding / nucleotide binding / mRNA binding / regulation of transcription by RNA polymerase II / nucleolus / negative regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II / mitochondrion / DNA binding / zinc ion binding / nucleoplasm / metal ion binding / nucleus / cytoplasm
Similarity search - Function
Mediator of RNA polymerase II transcription subunit 22, Saccharomycetes / Mediator of RNA polymerase II transcription subunit 20 / Mediator complex, subunit Med6, fungi / Mediator complex, subunit Med20 / TATA-binding related factor (TRF) of subunit 20 of Mediator complex / Mediator complex, subunit Med6 / Mediator complex, subunit Med17 / Mediator complex, subunit Med6 superfamily / MED6 mediator sub complex component / Subunit 17 of Mediator complex ...Mediator of RNA polymerase II transcription subunit 22, Saccharomycetes / Mediator of RNA polymerase II transcription subunit 20 / Mediator complex, subunit Med6, fungi / Mediator complex, subunit Med20 / TATA-binding related factor (TRF) of subunit 20 of Mediator complex / Mediator complex, subunit Med6 / Mediator complex, subunit Med17 / Mediator complex, subunit Med6 superfamily / MED6 mediator sub complex component / Subunit 17 of Mediator complex / Mediator of RNA polymerase II transcription subunit 22 / Mediator complex, subunit Med8, fungi/metazoa / Mediator complex, subunit Med11 / Surfeit locus protein 5 subunit 22 of Mediator complex / Mediator of RNA polymerase II transcription complex subunit 8 / Mediator complex protein / Mediator complex, subunit Med18 / Med18 protein / DNA-directed RNA polymerase II subunit Rpb4-like / RNA polymerase Rpb1 C-terminal repeat / RNA polymerase II, heptapeptide repeat, eukaryotic / Eukaryotic RNA polymerase II heptapeptide repeat. / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb4/RPC9, core / DNA-directed RNA-polymerase II subunit / 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. / RNA polymerase Rpb7-like , N-terminal / RNA polymerase Rpb7-like, N-terminal domain superfamily / RNA polymerase subunit Rpb7-like / SHS2 domain found in N terminus of Rpb7p/Rpc25p/MJ0397 / HRDC-like superfamily / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / 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 / DNA-directed RNA polymerases I, II, and III subunit RPABC4 / 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 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, 7 kDa subunit / RNA polymerase archaeal subunit P/eukaryotic subunit RPABC4 / RNA polymerase, subunit H/Rpb5, conserved site / RNA polymerases H / 23 Kd subunits signature. / RNA polymerase subunit CX / 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 Rpb11, 13-16kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo11 / RNA polymerases L / 13 to 16 Kd subunits signature. / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / Zinc finger, TFIIS-type / Transcription factor S-II (TFIIS) / Zinc finger TFIIS-type profile. / C2C2 Zinc finger / DNA-directed RNA polymerase, RBP11-like dimerisation domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase, subunit H/Rpb5 C-terminal / DNA-directed RNA polymerase subunit Rpo5/Rpb5 / 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. / Ribosomal protein S1-like RNA-binding domain / S1 RNA binding domain / S1 domain / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, insert domain
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 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 / Mediator of RNA polymerase II transcription subunit 17 / Mediator of RNA polymerase II transcription subunit 22 / Mediator of RNA polymerase II transcription subunit 18 / DNA-directed RNA polymerase II subunit RPB7 / Mediator of RNA polymerase II transcription subunit 20 / Mediator of RNA polymerase II transcription subunit 8 / Mediator of RNA polymerase II transcription subunit 6 / DNA-directed RNA polymerase II subunit RPB11 / DNA-directed RNA polymerases I, II, and III subunit RPABC4 / Mediator of RNA polymerase II transcription subunit 11
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 16.0 Å
AuthorsCai G / Chaban Y / Imasaki T / Kovacs JA / Calero G / Penczek PA / Takagi Y / Asturias FJ
CitationJournal: Structure / Year: 2012
Title: Interaction of the mediator head module with RNA polymerase II.
Authors: Gang Cai / Yuriy L Chaban / Tsuyoshi Imasaki / Julio A Kovacs / Guillermo Calero / Pawel A Penczek / Yuichiro Takagi / Francisco J Asturias /
Abstract: Mediator, a large (21 polypeptides, MW ∼1 MDa) complex conserved throughout eukaryotes, plays an essential role in control of gene expression by conveying regulatory signals that influence the ...Mediator, a large (21 polypeptides, MW ∼1 MDa) complex conserved throughout eukaryotes, plays an essential role in control of gene expression by conveying regulatory signals that influence the activity of the preinitiation complex. However, the precise mode of interaction between Mediator and RNA polymerase II (RNAPII), and the mechanism of regulation by Mediator remain elusive. We used cryo-electron microscopy and reconstituted in vitro transcription assays to characterize a transcriptionally-active complex including the Mediator Head module and components of a minimum preinitiation complex (RNAPII, TFIIF, TFIIB, TBP, and promoter DNA). Our results reveal how the Head interacts with RNAPII, affecting its conformation and function.
History
DepositionMar 6, 2012-
Header (metadata) releaseApr 5, 2012-
Map releaseMay 15, 2012-
UpdateMay 15, 2012-
Current statusMay 15, 2012Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.25
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 3.25
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j1n, PDB-3j1o
  • Surface level: 3.25
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3j1n
  • Imaged by Jmol
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3j1o
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5407_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5407.map.gz / Format: CCP4 / Size: 1.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of a complex including RNA polymerase II, Mediator Head module, TFIIF, TFIIB, TBP, and promoter DNA. TFIIB, TBP, and DNA are not visible in the map and are presumed to be disordered
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.83 Å/pix.
x 70 pix.
= 268.1 Å		3.83 Å/pix.
x 70 pix.
= 268.1 Å		3.83 Å/pix.
x 70 pix.
= 268.1 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.25 / Movie #1: 3.25
Minimum - Maximum-4.34038925 - 32.475795750000003
Average (Standard dev.)0.5565064 (±2.3740778)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions707070
Spacing707070
CellA=B=C: 268.1 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.833.833.83
M x/y/z707070
origin x/y/z0.0000.0000.000
length x/y/z268.100268.100268.100
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/NS707070
D min/max/mean-4.34032.4760.557

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Supplemental data

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Sample components

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Entire : Complex of RNA polymerase II, Mediator Head module, TFIIF, TFIIB,...

EntireName: Complex of RNA polymerase II, Mediator Head module, TFIIF, TFIIB, TBP, and promoter DNA
Components
  • Sample: Complex of RNA polymerase II, Mediator Head module, TFIIF, TFIIB, TBP, and promoter DNA
  • Protein or peptide: RNA polymerase II
  • Protein or peptide: Head module
  • Protein or peptide: TFIIF

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Supramolecule #1000: Complex of RNA polymerase II, Mediator Head module, TFIIF, TFIIB,...

SupramoleculeName: Complex of RNA polymerase II, Mediator Head module, TFIIF, TFIIB, TBP, and promoter DNA
type: sample / ID: 1000
Details: Although biochemical and functional evidence indicates the presence of all 6 original components (RNA polymerase II, Mediator Head, TFIIF, TFIIB, TBP, DNA), only 3 components (RNA polymerase ...Details: Although biochemical and functional evidence indicates the presence of all 6 original components (RNA polymerase II, Mediator Head, TFIIF, TFIIB, TBP, DNA), only 3 components (RNA polymerase II, Mediator Head, TFIIF) can be clearly identified in the cryo-EM map.
Number unique components: 6
Molecular weightTheoretical: 900 KDa / Method: sequence

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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 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: yeast
Molecular weightTheoretical: 540 KDa

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Macromolecule #2: Head module

MacromoleculeName: Head module / type: protein_or_peptide / ID: 2 / Name.synonym: Head / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: yeast
Molecular weightTheoretical: 240 KDa
Recombinant expressionOrganism: unidentified baculovirus / Recombinant plasmid: pBakPAC

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Macromolecule #3: TFIIF

MacromoleculeName: TFIIF / type: protein_or_peptide / ID: 3 / Name.synonym: IIF / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: yeast
Molecular weightTheoretical: 150 KDa

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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

Concentration0.3 mg/mL
BufferpH: 8 / Details: 25 mM KCl, 25 mM Tris-HCl, 10 mM DTT
GridDetails: 400 mesh Cu/Rh grids, coated with a perforated carbon film and glow discharged in the presence of amylamine
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 77 K / Instrument: HOMEMADE PLUNGER / Method: Blot for ~2 sec before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureMin: 77 K / Max: 120 K / Average: 115 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 125,000X magnification
DateJul 30, 2008
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Number real images: 500 / Average electron dose: 10 e/Å2 / Bits/pixel: 8
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 3.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each CCD frame
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Sparx,Spider / Number images used: 51000

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Atomic model buiding 1

Initial modelPDB ID:

1wcm


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E / Chain - #5 - Chain ID: F / Chain - #6 - Chain ID: G / Chain - #7 - Chain ID: H / Chain - #8 - Chain ID: I / Chain - #9 - Chain ID: J / Chain - #10 - Chain ID: K / Chain - #11 - Chain ID: L
SoftwareName: Monte Carlo Docking Analysis
DetailsProtocol: Rigid body fitting of individual RNA polymerase II structural modules. Fitted individual structural modules previously identified by X-ray crystallographic studies of RNA polymerase II under different crystallization conditions
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3j1n:
Cryo-EM map of a yeast minimal preinitiation complex interacting with the Mediator Head module

PDB-3j1o:
Cryo-EM map of a yeast minimal preinitiation complex interacting with the Mediator Head module

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Atomic model buiding 2

Initial modelPDB ID:

3rj1


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E / Chain - #5 - Chain ID: F / Chain - #6 - Chain ID: G
SoftwareName: Monte Carlo Docking Analysis
DetailsProtocol: Rigid body fitting of individual Mediator Head structural modules. Fitted individual structural modules previously identified by EM and X-ray crystallographic studies of the Head module
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3j1n:
Cryo-EM map of a yeast minimal preinitiation complex interacting with the Mediator Head module

PDB-3j1o:
Cryo-EM map of a yeast minimal preinitiation complex interacting with the Mediator Head module

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