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- EMDB-2193: human RNA polymerase II in complex with B1 RNA -

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Basic information

Entry
Database: EMDB / ID: EMD-2193
Titlehuman RNA polymerase II in complex with B1 RNA
Map datahuman RNA polymerase II in complex with B1 RNA
Sample
  • Sample: human RNA polymerase II in complex with B1 RNA
  • Protein or peptide: x 12 types
  • RNA: x 1 types
Keywordstranscription regulation / RNA polymerase II / non-coding RNA
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 25.0 Å
AuthorsKassube SA / Fang J / Grob P / Yakovchuk P / Goodrich JA / Nogales E
CitationJournal: J Mol Biol / Year: 2013
Title: Structural insights into transcriptional repression by noncoding RNAs that bind to human Pol II.
Authors: Susanne A Kassube / Jie Fang / Patricia Grob / Petro Yakovchuk / James A Goodrich / Eva Nogales /
Abstract: Gene transcription is regulated in response to environmental changes and developmental cues. In mammalian cells subjected to stress conditions such as heat shock, transcription of most protein-coding ...Gene transcription is regulated in response to environmental changes and developmental cues. In mammalian cells subjected to stress conditions such as heat shock, transcription of most protein-coding genes decreases, while the transcription of heat shock protein genes increases. Repression involves direct binding to RNA polymerase II (Pol II) of certain noncoding RNAs (ncRNAs) that are upregulated upon heat shock. Another class of ncRNAs is also upregulated and binds to Pol II but does not inhibit transcription. Incorporation of repressive ncRNAs into pre-initiation complexes prevents transcription initiation, while non-repressive ncRNAs are displaced from Pol II by TFIIF. Here, we present cryo-electron microscopy reconstructions of human Pol II in complex with six different ncRNAs from mouse and human. Our structures show that both repressive and non-repressive ncRNAs bind to a conserved binding site within the cleft of Pol II. The site, which is also shared with a previously characterized yeast aptamer, is close to the active center and, thus, in an ideal position to regulate transcription. Importantly, additional RNA elements extend flexibly beyond the docking site. We propose that the differences concerning the repressive activity of the ncRNAs analyzed must be due to the distinct character of these more unstructured, flexible segments of the RNA that emanate from the cleft.
History
DepositionSep 11, 2012-
Header (metadata) releaseSep 26, 2012-
Map releaseJan 16, 2013-
UpdateSep 18, 2013-
Current statusSep 18, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.085
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.085
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

image2193.png

Downloads & links

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Map

FileDownload / File: emd_2193.map.gz / Format: CCP4 / Size: 11.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationhuman RNA polymerase II in complex with B1 RNA
Voxel sizeX=Y=Z: 2.54 Å
Density
Contour LevelBy AUTHOR: 0.085 / Movie #1: 0.085
Minimum - Maximum-0.05299269 - 0.17365298
Average (Standard dev.)-0.0013166 (±0.01599781)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions146146146
Spacing146146146
CellA=B=C: 370.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.542.542.54
M x/y/z146146146
origin x/y/z0.0000.0000.000
length x/y/z370.840370.840370.840
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS146146146
D min/max/mean-0.0530.174-0.001

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Supplemental data

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Sample components

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Entire : human RNA polymerase II in complex with B1 RNA

EntireName: human RNA polymerase II in complex with B1 RNA
Components
  • Sample: human RNA polymerase II in complex with B1 RNA
  • Protein or peptide: Rpb12
  • Protein or peptide: Rpb11
  • Protein or peptide: Rpb10
  • Protein or peptide: Rpb9
  • Protein or peptide: Rpb8
  • Protein or peptide: Rpb7
  • Protein or peptide: Rpb6POLR2F
  • Protein or peptide: Rpb5
  • Protein or peptide: Rpb4
  • Protein or peptide: Rpb3
  • Protein or peptide: Rpb2POLR2B
  • Protein or peptide: Rpb1POLR2A
  • RNA: B1 RNA

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Supramolecule #1000: human RNA polymerase II in complex with B1 RNA

SupramoleculeName: human RNA polymerase II in complex with B1 RNA / type: sample / ID: 1000
Oligomeric state: RNA polymerase II (12 polypeptide chains) binds to B1 RNA
Number unique components: 13
Molecular weightTheoretical: 520 KDa

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Macromolecule #1: Rpb12

MacromoleculeName: Rpb12 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 10 KDa

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Macromolecule #3: Rpb11

MacromoleculeName: Rpb11 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 10 KDa

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Macromolecule #4: Rpb10

MacromoleculeName: Rpb10 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 10 KDa

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Macromolecule #5: Rpb9

MacromoleculeName: Rpb9 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 10 KDa

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Macromolecule #6: Rpb8

MacromoleculeName: Rpb8 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 20 KDa

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Macromolecule #7: Rpb7

MacromoleculeName: Rpb7 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 20 KDa

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Macromolecule #8: Rpb6

MacromoleculeName: Rpb6 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 10 KDa

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Macromolecule #9: Rpb5

MacromoleculeName: Rpb5 / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 20 KDa

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Macromolecule #10: Rpb4

MacromoleculeName: Rpb4 / type: protein_or_peptide / ID: 10 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 20 KDa

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Macromolecule #11: Rpb3

MacromoleculeName: Rpb3 / type: protein_or_peptide / ID: 11 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 30 KDa

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Macromolecule #12: Rpb2

MacromoleculeName: Rpb2 / type: protein_or_peptide / ID: 12 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 130 KDa

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Macromolecule #13: Rpb1

MacromoleculeName: Rpb1 / type: protein_or_peptide / ID: 13 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: HeLa / Cell: HeLa / Organelle: Nucleus / Location in cell: Nucleus
Molecular weightTheoretical: 220 KDa

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Macromolecule #2: B1 RNA

MacromoleculeName: B1 RNA / type: rna / ID: 2 / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: No
Source (natural)Organism: Mus musculus (house mouse) / synonym: Mouse
Molecular weightTheoretical: 45 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

Concentration0.0312 mg/mL
BufferpH: 7.9
Details: 10 mM Tris pH 7.9, 10 mM HEPES pH 8.0, 4 mM MgCl2, 50 mM KCl, 0.05% NP-40, 1 mM DTT, 0.1% trehalose
GridDetails: 400 mesh copper grids covered with a holey carbon film, thin carbon film floated on top
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 86 K / Instrument: FEI VITROBOT MARK II / Method: 6 sec blot, offset -2

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50280 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 50000
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
DateOct 6, 2008
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: 133 / Average electron dose: 20 e/Å2 / Od range: 1 / Bits/pixel: 14

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Image processing

CTF correctionDetails: whole micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2, FREALIGN
Details: Projection matching using a low-pass filtered model of apo Pol II as the starting model
Number images used: 25079

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