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Yorodumi- EMDB-2367: Structural mimicry in transcription regulation of human RNA polym... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2367 | |||||||||
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Title | Structural mimicry in transcription regulation of human RNA polymerase II by the DNA helicase RECQL5 | |||||||||
Map data | Reconstruction of elongating human Pol II in complex with the DNA helicase RECQL5 | |||||||||
Sample |
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Keywords | transcription regulation / RNA polymerase II / RecQ helicase / RECQL5 / DNA repair | |||||||||
Biological species | Homo sapiens (human) / unidentified (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 13.0 Å | |||||||||
Authors | Kassube SA / Jinek M / Fang J / Tsutakawa S / Nogales E | |||||||||
Citation | Journal: Nat Struct Mol Biol / Year: 2013 Title: Structural mimicry in transcription regulation of human RNA polymerase II by the DNA helicase RECQL5. Authors: Susanne A Kassube / Martin Jinek / Jie Fang / Susan Tsutakawa / Eva Nogales / Abstract: RECQL5 is a member of the highly conserved RecQ family of DNA helicases involved in DNA repair. RECQL5 interacts with RNA polymerase II (Pol II) and inhibits transcription of protein-encoding genes ...RECQL5 is a member of the highly conserved RecQ family of DNA helicases involved in DNA repair. RECQL5 interacts with RNA polymerase II (Pol II) and inhibits transcription of protein-encoding genes by an unknown mechanism. We show that RECQL5 contacts the Rpb1 jaw domain of Pol II at a site that overlaps with the binding site for the transcription elongation factor TFIIS. Our cryo-EM structure of elongating Pol II arrested in complex with RECQL5 shows that the RECQL5 helicase domain is positioned to sterically block elongation. The crystal structure of the RECQL5 KIX domain reveals similarities with TFIIS, and binding of RECQL5 to Pol II interferes with the ability of TFIIS to promote transcriptional read-through in vitro. Together, our findings reveal a dual mode of transcriptional repression by RECQL5 that includes structural mimicry of the Pol II-TFIIS interaction. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_2367.map.gz | 1 MB | EMDB map data format | |
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Header (meta data) | emd-2367-v30.xml emd-2367.xml | 13.2 KB 13.2 KB | Display Display | EMDB header |
Images | EMD-2367-RECQL5_skassube.jpg | 686.4 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2367 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2367 | HTTPS FTP |
-Validation report
Summary document | emd_2367_validation.pdf.gz | 197 KB | Display | EMDB validaton report |
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Full document | emd_2367_full_validation.pdf.gz | 196.1 KB | Display | |
Data in XML | emd_2367_validation.xml.gz | 5.7 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2367 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2367 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_2367.map.gz / Format: CCP4 / Size: 20.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of elongating human Pol II in complex with the DNA helicase RECQL5 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.31 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Human Pol II in complex with artificial transcription bubble and ...
Entire | Name: Human Pol II in complex with artificial transcription bubble and RECQL5 |
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Components |
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-Supramolecule #1000: Human Pol II in complex with artificial transcription bubble and ...
Supramolecule | Name: Human Pol II in complex with artificial transcription bubble and RECQL5 type: sample / ID: 1000 Oligomeric state: 12-subunit Pol II complex bound to transcription bubble and monomeric RECQL5 Number unique components: 5 |
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Molecular weight | Theoretical: 615 KDa |
-Macromolecule #1: RNA polymerase II
Macromolecule | Name: RNA polymerase II / type: protein_or_peptide / ID: 1 / Name.synonym: Pol II / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No |
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Source (natural) | Organism: Homo sapiens (human) / Strain: HeLa / synonym: Human / Organelle: Nucleus |
Molecular weight | Theoretical: 517 KDa |
-Macromolecule #5: RECQL5
Macromolecule | Name: RECQL5 / type: protein_or_peptide / ID: 5 / Name.synonym: RecQ5 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human |
Molecular weight | Theoretical: 69.3 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant plasmid: pGEX-6P-1 |
-Macromolecule #2: transcription bubble RNA strand
Macromolecule | Name: transcription bubble RNA strand / type: rna / ID: 2 / Classification: OTHER / Structure: OTHER / Synthetic?: No |
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Source (natural) | Organism: unidentified (others) |
Molecular weight | Theoretical: 6.1 KDa |
Sequence | String: UAUAUGCAUA AAGACCAGGC |
-Macromolecule #3: non-template strand
Macromolecule | Name: non-template strand / type: dna / ID: 3 / Classification: DNA / Structure: OTHER / Synthetic?: No |
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Source (natural) | Organism: unidentified (others) |
Molecular weight | Theoretical: 13.3 KDa |
Sequence | String: TAGTAAACTA GTATTGAAAG TACTTGAGCT TAGACAGCAT GTC |
-Macromolecule #4: template strand
Macromolecule | Name: template strand / type: dna / ID: 4 / Classification: DNA / Structure: OTHER / Synthetic?: No |
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Source (natural) | Organism: unidentified (others) |
Molecular weight | Theoretical: 8.5 KDa |
Sequence | String: CTCAAGTACT TACGCCTGGT CATTACTA |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.037 mg/mL |
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Buffer | pH: 8 Details: 20 mM Hepes pH 8.0, 4mM MgCl2, 50 mM KCl, 0.05% NP-40, 1mM TCEP |
Grid | Details: 400-mesh C-flats (Protochips Inc.) with thin carbon support (Protochips Inc.) |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 86 K / Instrument: FEI VITROBOT MARK II / Method: blot for 2 sec, 0 offset |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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Temperature | Average: 78 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification Legacy - Electron beam tilt params: 0 |
Date | Oct 6, 2012 |
Image recording | Category: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Number real images: 5224 / Average electron dose: 20 e/Å2 |
Electron beam | Acceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal defocus max: 2.8 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 100000 |
Sample stage | Specimen holder: Side-entry cryostage / Specimen holder model: GATAN LIQUID NITROGEN |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-Image processing
Details | Image processing was performed in the Appion processing environment. 3D reconstruction was performed using EMAN2 and SPARX libraries. Final map was filtered to local resolution using the blocres function of the Bsoft package. |
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CTF correction | Details: whole micrograph |
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2, SPARX / Number images used: 121416 |
-Atomic model buiding 1
Initial model | PDB ID: |
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Software | Name: Chimera |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
-Atomic model buiding 2
Initial model | PDB ID: |
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Software | Name: Chimera |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
-Atomic model buiding 3
Initial model | PDB ID: |
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Refinement | Space: REAL / Protocol: RIGID BODY FIT |