+Open data
-Basic information
Entry | Database: PDB / ID: 7rqs | |||||||||
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Title | Arabidopsis RNA-dependent RNA polymerase 2 | |||||||||
Components | RNA-dependent RNA polymerase 2 | |||||||||
Keywords | TRANSCRIPTION / siRNA / epigenetics / plants / RNA polymerase / gene silencing | |||||||||
Function / homology | Function and homology information siRNA-mediated long-distance post-transcriptional gene silencing / siRNA transcription / DNA/RNA hybrid binding / siRNA processing / defense response to fungus / single-stranded RNA binding / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / nucleolus / nucleoplasm ...siRNA-mediated long-distance post-transcriptional gene silencing / siRNA transcription / DNA/RNA hybrid binding / siRNA processing / defense response to fungus / single-stranded RNA binding / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / nucleolus / nucleoplasm / metal ion binding / nucleus Similarity search - Function | |||||||||
Biological species | Arabidopsis thaliana (thale cress) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.57 Å | |||||||||
Authors | Fukudome, A. / Pikaard, C.S. / Takagi, Y. | |||||||||
Funding support | United States, 2items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2021 Title: Structure and RNA template requirements of RNA-DEPENDENT RNA POLYMERASE 2. Authors: Akihito Fukudome / Jasleen Singh / Vibhor Mishra / Eswar Reddem / Francisco Martinez-Marquez / Sabine Wenzel / Rui Yan / Momoko Shiozaki / Zhiheng Yu / Joseph Che-Yen Wang / Yuichiro Takagi / Craig S Pikaard / Abstract: RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In , RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA ...RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In , RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest, induced by the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The double-stranded RNAs are then released from the Pol IV-RDR2 complex and diced into short-interfering RNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 Å, determined by cryoelectron microscopy. The N-terminal region contains an RNA-recognition motif adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1 to 2 nt internal to the 3' ends of its templates and can transcribe the RNA of an RNA/DNA hybrid, provided that 9 or more nucleotides are unpaired at the RNA's 3' end. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3' end occurs as the DNA template and nontemplate strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3' end as the DNA strands reanneal, allowing RDR2 to engage the RNA and synthesize the complementary strand. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7rqs.cif.gz | 193.7 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7rqs.ent.gz | 145.1 KB | Display | PDB format |
PDBx/mmJSON format | 7rqs.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/rq/7rqs ftp://data.pdbj.org/pub/pdb/validation_reports/rq/7rqs | HTTPS FTP |
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-Related structure data
Related structure data | 24635MC 7rozC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 133619.828 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: RDR2, RDRP2, SMD1, At4g11130, F2P3.11 Production host: Insect cell expression vector pTIE1 (others) References: UniProt: O82504, RNA-directed RNA polymerase |
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#2: Chemical | ChemComp-MG / |
Has ligand of interest | Y |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: RNA-dependent RNA polymerase 2 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT |
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Molecular weight | Value: 0.129 MDa / Experimental value: NO |
Source (natural) | Organism: Arabidopsis thaliana (thale cress) |
Source (recombinant) | Organism: Insect cell expression vector pTIE1 (others) |
Buffer solution | pH: 7.6 |
Specimen | Conc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample was mono disperse. |
Specimen support | Grid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: UltrAuFoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company | |||||||||
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Microscopy | Model: FEI TITAN KRIOS | |||||||||
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER | |||||||||
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 59242 X / Nominal defocus max: -2000 nm / Nominal defocus min: -800 nm / Cs: 2.7 mm | |||||||||
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER | |||||||||
Image recording | Imaging-ID: 1 / Average exposure time: 4 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1
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-Processing
EM software |
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CTF correction | Type: NONE | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.57 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 277019 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 173.2 / Protocol: AB INITIO MODEL / Space: REAL |