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- EMDB-24610: Structure of RNA-dependent RNA polymerase 2 (RDR2) from Arabidops... -

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Basic information

Entry
Database: EMDB / ID: EMD-24610
TitleStructure of RNA-dependent RNA polymerase 2 (RDR2) from Arabidopsis thaliana
Map dataEM map for RNA dependent RNA polymerase 2 from Arabidopsis
Sample
  • Complex: RNA-dependent RNA polymerase 2 (RDR2)
    • Protein or peptide: RNA-dependent RNA polymerase 2
  • Ligand: MAGNESIUM ION
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsFukudome A / Pikaard CS / Takagi Y
Funding support United States, 2 items
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, 2021-
Header (metadata) releaseDec 8, 2021-
Map releaseDec 8, 2021-
UpdateJun 22, 2022-
Current statusJun 22, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0138
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0138
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7roz
  • Surface level: 0.0138
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24610.png

Downloads & links

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Map

FileDownload / File: emd_24610.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEM map for RNA dependent RNA polymerase 2 from Arabidopsis
Voxel sizeX=Y=Z: 0.844 Å
Density
Contour LevelBy AUTHOR: 0.0138 / Movie #1: 0.0138
Minimum - Maximum-0.025013156 - 0.056713667
Average (Standard dev.)8.890075e-06 (±0.0013016883)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 270.08 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8440.8440.844
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z270.080270.080270.080
α/β/γ90.00090.00090.000
start NX/NY/NZ106102101
NX/NY/NZ87104102
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0250.0570.000

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Supplemental data

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Sample components

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Entire : RNA-dependent RNA polymerase 2 (RDR2)

EntireName: RNA-dependent RNA polymerase 2 (RDR2)
Components
  • Complex: RNA-dependent RNA polymerase 2 (RDR2)
    • Protein or peptide: RNA-dependent RNA polymerase 2
  • Ligand: MAGNESIUM ION

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Supramolecule #1: RNA-dependent RNA polymerase 2 (RDR2)

SupramoleculeName: RNA-dependent RNA polymerase 2 (RDR2) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Recombinant expressionOrganism: Insect cell expression vector pTIE1 (others)
Molecular weightTheoretical: 129 KDa

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Macromolecule #1: RNA-dependent RNA polymerase 2

MacromoleculeName: RNA-dependent RNA polymerase 2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA-directed RNA polymerase
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 133.619828 KDa
Recombinant expressionOrganism: Insect cell expression vector pTIE1 (others)
SequenceString: MVSETTTNRS TVKISNVPQT IVADELLRFL ELHLGEDTVF ALEIPTTRDN WKPRDFARVQ FTTLEVKSRA QLLSSQSKLL FKTHNLRLS EAYDDIIPRP VDPRKRLDDI VLTVGFPESD EKRFCALEKW DGVRCWILTE KRRVEFWVWE SGDCYKIEVR F EDIIETLS ...String:
MVSETTTNRS TVKISNVPQT IVADELLRFL ELHLGEDTVF ALEIPTTRDN WKPRDFARVQ FTTLEVKSRA QLLSSQSKLL FKTHNLRLS EAYDDIIPRP VDPRKRLDDI VLTVGFPESD EKRFCALEKW DGVRCWILTE KRRVEFWVWE SGDCYKIEVR F EDIIETLS CCVNGDASEI DAFLLKLKYG PKVFKRVTVH IATKFKSDRY RFCKEDFDFM WIRTTDFSGS KSIGTSTCFC LE VHNGSTM LDIFSGLPYY REDTLSLTYV DGKTFASAAQ IVPLLNAAIL GLEFPYEILF QLNALVHAQK ISLFAASDME LIK ILRGMS LETALVILKK LHQQSSICYD PVFFVKTQMQ SVVKKMKHSP ASAYKRLTEQ NIMSCQRAYV TPSKIYLLGP ELET ANYVV KNFAEHVSDF MRVTFVEEDW SKLPANALSV NSKEGYFVKP SRTNIYNRVL SILGEGITVG PKRFEFLAFS ASQLR GNSV WMFASNEKVK AEDIREWMGC FRKIRSISKC AARMGQLFSA SRQTLIVRAQ DVEQIPDIEV TTDGADYCFS DGIGKI SLA FAKQVAQKCG LSHVPSAFQI RYGGYKGVIA VDRSSFRKLS LRDSMLKFDS NNRMLNVTRW TESMPCFLNR EIICLLS TL GIEDAMFEAM QAVHLSMLGN MLEDRDAALN VLQKLSGENS KNLLVKMLLQ GYAPSSEPYL SMMLRVHHES QLSELKSR C RILVPKGRIL IGCMDEMGIL EYGQVYVRVT LTKAELKSRD QSYFRKIDEE TSVVIGKVVV TKNPCLHPGD IRVLDAIYE VHFEEKGYLD CIIFPQKGER PHPNECSGGD LDGDQFFVSW DEKIIPSEMD PPMDYAGSRP RLMDHDVTLE EIHKFFVDYM ISDTLGVIS TAHLVHADRD PEKARSQKCL ELANLHSRAV DFAKTGAPAE MPYALKPREF PDFLERFEKP TYISESVFGK L YRAVKSSL AQRKPEAESE DTVAYDVTLE EAGFESFIET AKAHRDMYGE KLTSLMIYYG AANEEEILTG ILKTKEMYLA RD NRRYGDM KDRITLSVKD LHKEAMGWFE KSCEDEQQKK KLASAWYYVT YNPNHRDEKL TFLSFPWIVG DVLLDIKAEN AQR QSVEEK TSGLVSIKLE NLYFQGSAWS HPQFEKGGGS GGGSGGSAWS HPQFEK

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration0.7 mg/mL
BufferpH: 7.6
GridPretreatment - Type: GLOW DISCHARGE / Details: unspecified
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
DetailsThe sample was mono disperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 59242 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: -2.0 µm / Nominal defocus min: -0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recording#0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / #0 - Number grids imaged: 1 / #0 - Number real images: 6417 / #0 - Average exposure time: 4.0 sec. / #0 - Average electron dose: 60.0 e/Å2 / #0 - Details: Non-tilted dataset / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / #1 - Number grids imaged: 1 / #1 - Number real images: 4545 / #1 - Average exposure time: 4.0 sec. / #1 - Average electron dose: 60.0 e/Å2 / #1 - Details: 30 degree titled dataset
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 118561
Image recording ID1
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 73.71
Output model

PDB-7roz:
Structure of RNA-dependent RNA polymerase 2 (RDR2) from Arabidopsis thaliana

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