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- PDB-7roz: Structure of RNA-dependent RNA polymerase 2 (RDR2) from Arabidops... -

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Basic information

Entry
Database: PDB / ID: 7roz
TitleStructure of RNA-dependent RNA polymerase 2 (RDR2) from Arabidopsis thaliana
ComponentsRNA-dependent RNA polymerase 2
KeywordsTRANSCRIPTION / 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
RNA-dependent RNA polymerase, eukaryotic-type / RNA dependent RNA polymerase
Similarity search - Domain/homology
RNA-dependent RNA polymerase 2
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsFukudome, A. / Pikaard, C.S. / Takagi, Y.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R01GM111695 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R01GM077590 United States
CitationJournal: 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
DepositionAug 2, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 8, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 22, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

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Assembly

Deposited unit
A: RNA-dependent RNA polymerase 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)133,6442
Polymers133,6201
Non-polymers241
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein RNA-dependent RNA polymerase 2 / / AtRDRP2 / Protein SILENCING MOVEMENT DEFICIENT 1 / RNA-directed RNA polymerase 2


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
#2: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: RNA-dependent RNA polymerase 2 (RDR2) / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.129 MDa / Experimental value: NO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Insect cell expression vector pTIE1 (others)
Buffer solutionpH: 7.6
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample was mono disperse.
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 59242 X / Nominal defocus max: -2000 nm / Nominal defocus min: -800 nm / Cs: 2.7 mm
Specimen holderCryogen: 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

IDNum. of real imagesDetails
16417Non-tilted dataset
2454530 degree titled dataset

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Processing

EM software
IDNameVersionCategory
7Buccaneermodel fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
12RELION3.13D reconstruction
13PHENIXmodel refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 118561 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 73.71 / Protocol: AB INITIO MODEL / Space: REAL

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