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- PDB-7eu1: The cryo-EM structure of A. thaliana Pol IV-RDR2 holoenzyme -

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Basic information

Entry
Database: PDB / ID: 7eu1
TitleThe cryo-EM structure of A. thaliana Pol IV-RDR2 holoenzyme
Components
  • (DNA-directed RNA polymerases ...RNA polymerase) x 9
  • DNA-directed RNA polymerase IV subunit 1Polymerase
  • RNA-dependent RNA polymerase 2
KeywordsTRANSCRIPTION / DNA-directed RNA polymerase IV / RNA- dependent RNA polymerase 2 / RNA-directed DNA methylation pathway / TRANSPORT PROTEIN
Function / homology
Function and homology information


RNA polymerase IV complex / stomatal complex patterning / siRNA-mediated long-distance post-transcriptional gene silencing / retrotransposon silencing by siRNA-directed DNA methylation / RNA polymerase V complex / stomatal complex development / siRNA transcription / DNA/RNA hybrid binding / regulatory ncRNA-mediated post-transcriptional gene silencing / : ...RNA polymerase IV complex / stomatal complex patterning / siRNA-mediated long-distance post-transcriptional gene silencing / retrotransposon silencing by siRNA-directed DNA methylation / RNA polymerase V complex / stomatal complex development / siRNA transcription / DNA/RNA hybrid binding / regulatory ncRNA-mediated post-transcriptional gene silencing / : / siRNA processing / regulatory ncRNA-mediated gene silencing / RNA polymerase complex / RNA polymerase III activity / regulation of immune response / RNA polymerase I activity / RNA polymerase I complex / RNA polymerase III complex / RNA polymerase II activity / defense response to fungus / heterochromatin / RNA polymerase II, core complex / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / nucleic acid binding / single-stranded RNA binding / protein dimerization activity / RNA-directed RNA polymerase / RNA-dependent RNA polymerase activity / DNA-templated transcription / nucleolus / DNA binding / zinc ion binding / nucleoplasm / metal ion binding / nucleus / cytoplasm
Similarity search - Function
RNA-dependent RNA polymerase, eukaryotic-type / RNA dependent RNA polymerase / Pol II subunit B9, C-terminal zinc ribbon / RNA polymerase RBP11 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / DNA-directed RNA polymerase, M/15kDa subunit / RNA polymerases M/15 Kd subunit / RNA polymerase subunit 9 / DNA-directed RNA polymerase subunit RPABC5/Rpb10 ...RNA-dependent RNA polymerase, eukaryotic-type / RNA dependent RNA polymerase / Pol II subunit B9, C-terminal zinc ribbon / RNA polymerase RBP11 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / DNA-directed RNA polymerase, M/15kDa subunit / RNA polymerases M/15 Kd subunit / RNA polymerase subunit 9 / DNA-directed RNA polymerase subunit RPABC5/Rpb10 / RNA polymerases, subunit N, zinc binding site / RNA polymerase subunit RPB10 / RNA polymerases N / 8 kDa subunit / RNA polymerases N / 8 Kd subunits signature. / DNA-directed RNA polymerase M, 15kDa subunit, conserved site / RNA polymerases M / 15 Kd subunits signature. / DNA-directed RNA polymerase subunit/transcription factor S / RNA polymerase, Rpb8 / DNA-directed RNA polymerases I, II, and III subunit RPABC4 / RNA polymerase Rpb8 / RNA polymerase subunit 8 / RNA polymerase, Rpb5, N-terminal / RNA polymerase Rpb5, N-terminal domain superfamily / RNA polymerase Rpb5, N-terminal domain / DNA-directed RNA polymerase, subunit RPB6 / DNA directed RNA polymerase, 7 kDa subunit / DNA-directed RNA polymerase subunit Rpo11 / RNA polymerase archaeal subunit P/eukaryotic subunit RPABC4 / RNA polymerase, subunit H/Rpb5, conserved site / RNA polymerases H / 23 Kd subunits signature. / RNA polymerase subunit CX / DNA-directed RNA polymerase Rpb11, 13-16kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo5/Rpb5 / RNA polymerases L / 13 to 16 Kd subunits signature. / DNA-directed RNA polymerase, 30-40kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo3/Rpb3/RPAC1 / RNA polymerases D / 30 to 40 Kd subunits signature. / DNA-directed RNA polymerase, RBP11-like dimerisation domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase, subunit H/Rpb5 C-terminal / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / RPB5-like RNA polymerase subunit superfamily / RNA polymerase Rpb5, C-terminal domain / Zinc finger, TFIIS-type / Transcription factor S-II (TFIIS) / Zinc finger TFIIS-type profile. / C2C2 Zinc finger / Archaeal Rpo6/eukaryotic RPB6 RNA polymerase subunit / DNA-directed RNA polymerase, 14-18kDa subunit, conserved site / RNA polymerases K / 14 to 18 Kd subunits signature. / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RPB6/omega subunit-like superfamily / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, OB-fold / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerases beta chain signature. / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 6 / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
DNA-directed RNA polymerases II and IV subunit 5A / RNA-dependent RNA polymerase 2 / DNA-directed RNA polymerases II, IV and V subunit 11 / DNA-directed RNA polymerases II, IV and V subunit 3 / DNA-directed RNA polymerases II, IV and V subunit 9A / DNA-directed RNA polymerases II, IV and V subunit 10 / DNA-directed RNA polymerases II, IV and V subunit 6A / DNA-directed RNA polymerases II, IV and V subunit 12 / DNA-directed RNA polymerases IV and V subunit 2 / DNA-directed RNA polymerases II, IV and V subunit 8B
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.86 Å
AuthorsFang, C.L. / Wu, X.X. / Huang, K. / Zhang, Y.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Science / Year: 2021
Title: Pol IV and RDR2: A two-RNA-polymerase machine that produces double-stranded RNA.
Authors: Kun Huang / Xiao-Xian Wu / Cheng-Li Fang / Zhou-Geng Xu / Hong-Wei Zhang / Jian Gao / Chuan-Miao Zhou / Lin-Lin You / Zhan-Xi Gu / Wen-Hui Mu / Yu Feng / Jia-Wei Wang / Yu Zhang /
Abstract: DNA methylation affects gene expression and maintains genome integrity. The DNA-dependent RNA polymerase IV (Pol IV), together with the RNA-dependent RNA polymerase RDR2, produces double-stranded ...DNA methylation affects gene expression and maintains genome integrity. The DNA-dependent RNA polymerase IV (Pol IV), together with the RNA-dependent RNA polymerase RDR2, produces double-stranded small interfering RNA precursors essential for establishing and maintaining DNA methylation in plants. We determined the cryo–electron microscopy structures of the Pol IV–RDR2 holoenzyme and the backtracked transcription elongation complex. These structures reveal that Pol IV and RDR2 form a complex with their active sites connected by an interpolymerase channel, through which the Pol IV–generated transcript is handed over to the RDR2 active site after being backtracked, where it is used as the template for double-stranded RNA (dsRNA) synthesis. Our results describe a ‘backtracking-triggered RNA channeling’ mechanism underlying dsRNA synthesis and also shed light on the evolutionary trajectory of eukaryotic RNA polymerases.
History
DepositionMay 15, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 29, 2021Provider: repository / Type: Initial release
Revision 1.1Feb 16, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed
Revision 1.2Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Assembly

Deposited unit
A: DNA-directed RNA polymerase IV subunit 1
B: DNA-directed RNA polymerases IV and V subunit 2
C: DNA-directed RNA polymerases II, IV and V subunit 3
E: DNA-directed RNA polymerases II and IV subunit 5A
F: DNA-directed RNA polymerases II, IV and V subunit 6A
H: DNA-directed RNA polymerases II, IV and V subunit 8B
I: DNA-directed RNA polymerases II, IV and V subunit 9A
J: DNA-directed RNA polymerases II, IV and V subunit 10
K: DNA-directed RNA polymerases II, IV and V subunit 11
L: DNA-directed RNA polymerases II, IV and V subunit 12
M: RNA-dependent RNA polymerase 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)566,69921
Polymers566,12711
Non-polymers57210
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules AM

#1: Protein DNA-directed RNA polymerase IV subunit 1 / Polymerase


Mass: 169535.281 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Arabidopsis thaliana (thale cress)
#11: Protein RNA-dependent RNA polymerase 2 / / AtRDRP2 / Protein SILENCING MOVEMENT DEFICIENT 1 / RNA-directed RNA polymerase 2


Mass: 129494.531 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Arabidopsis thaliana (thale cress) / References: UniProt: O82504, RNA-directed RNA polymerase

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DNA-directed RNA polymerases ... , 9 types, 9 molecules BCEFHIJKL

#2: Protein DNA-directed RNA polymerases IV and V subunit 2 / DNA-directed RNA polymerase D subunit 2a / AtNRPD2a / Nuclear RNA polymerase D 2a / Nuclear RNA ...DNA-directed RNA polymerase D subunit 2a / AtNRPD2a / Nuclear RNA polymerase D 2a / Nuclear RNA polymerase E 2 / Protein DEFECTIVE IN MERISTEM SILENCING 2 / Protein DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 2 / RNA polymerase IV subunit 2a / POL IV 2a


Mass: 132848.047 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Arabidopsis thaliana (thale cress) / References: UniProt: Q9LK40, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerases II, IV and V subunit 3 / RNA polymerase / DNA-directed RNA polymerase II 36 kDa polypeptide A / DNA-directed RNA polymerase II subunit RPB3-A ...DNA-directed RNA polymerase II 36 kDa polypeptide A / DNA-directed RNA polymerase II subunit RPB3-A / RNA polymerase II subunit 3-A / RNA polymerase II subunit B3-A


Mass: 35503.219 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Arabidopsis thaliana (thale cress) / References: UniProt: Q39211
#4: Protein DNA-directed RNA polymerases II and IV subunit 5A / RPB5a / DNA-directed RNA polymerase II subunit E / RNA polymerase I / II and III 24.3 kDa subunit / AtRPB24.3


Mass: 24340.389 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Arabidopsis thaliana (thale cress) / References: UniProt: O81098
#5: Protein DNA-directed RNA polymerases II, IV and V subunit 6A / RNA polymerase / RNA polymerase Rpb6


Mass: 16670.346 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Arabidopsis thaliana (thale cress) / References: UniProt: Q9FJ98
#6: Protein DNA-directed RNA polymerases II, IV and V subunit 8B / RNA polymerase / RNA polymerase Rpb8


Mass: 16626.299 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Arabidopsis thaliana (thale cress) / References: UniProt: Q9M1A8
#7: Protein DNA-directed RNA polymerases II, IV and V subunit 9A / RNA polymerase


Mass: 13297.145 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Arabidopsis thaliana (thale cress) / References: UniProt: Q6NLH0
#8: Protein DNA-directed RNA polymerases II, IV and V subunit 10 / RNA polymerase / DNA-directed RNA Polymerase II subunit L


Mass: 8323.690 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Arabidopsis thaliana (thale cress) / References: UniProt: Q8LFJ6
#9: Protein DNA-directed RNA polymerases II, IV and V subunit 11 / RNA polymerase / DNA-directed RNA polymerase II 13.6 kDa polypeptide / DNA-directed RNA polymerase II subunit J / ...DNA-directed RNA polymerase II 13.6 kDa polypeptide / DNA-directed RNA polymerase II subunit J / DNA-directed RNA polymerase II subunit RPB11 / RNA polymerase II subunit B11


Mass: 13582.286 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Arabidopsis thaliana (thale cress) / References: UniProt: Q38859
#10: Protein DNA-directed RNA polymerases II, IV and V subunit 12 / RNA polymerase / DNA-directed RNA Polymerase II subunit K


Mass: 5905.768 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Arabidopsis thaliana (thale cress) / References: UniProt: Q9FLM8

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Non-polymers , 2 types, 10 molecules

#12: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Zn
#13: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN

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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: A. thaliana Pol IV-RDR2 holoenzyme / Type: COMPLEX / Entity ID: #1-#11 / Source: NATURAL
Molecular weightUnits: KILODALTONS/NANOMETER / Experimental value: NO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mmol/LHEPESC2H18N2O4S1
2100 mmol/LSodium chlorideNaClSodium chloride1
35 mmol/LMagnesium chlorideMgCl21
42 mmol/LDL-dithiothreitolC4H10O2S21
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The complex contains 11 protein subunits (A, B, C, E, F, H , I, J, K, L, M)
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 282.65 K

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingAverage exposure time: 3.366 sec. / Electron dose: 58.8 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
4GctfCTF correction
9RELION3initial Euler assignment
10RELION3final Euler assignment
12RELION33D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 3.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 132247 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL

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