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- PDB-8him: A cryo-EM structure of B. oleracea RNA polymerase V elongation co... -

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Basic information

Entry
Database: PDB / ID: 8him
TitleA cryo-EM structure of B. oleracea RNA polymerase V elongation complex at 2.73 Angstrom
Components
  • (DNA (34-MER)) x 2
  • (DNA-directed RNA polymerase ...Polymerase) x 3
  • (DNA-directed RNA polymerases I, II, and III subunit ...RNA polymerase) x 4
  • DNA-directed RNA polymerases II, IV and V subunit 12RNA polymerase
  • RNA (5'-R(*UP*AP*UP*AP*UP*GP*CP*AP*UP*AP*AP*AP*GP*AP*CP*CP*AP*GP*GP*C)-3')
  • RNA_pol_L_2 domain-containing protein
  • RPOLD domain-containing protein
KeywordsTRANSCRIPTION / DNA-dependent RNA polymerase V
Function / homology
Function and homology information


: / RNA polymerase II activity / RNA polymerase I complex / RNA polymerase III complex / RNA polymerase II, core complex / DNA-directed RNA polymerase complex / DNA-directed 5'-3' RNA polymerase activity / nucleic acid binding / protein dimerization activity / DNA-templated transcription ...: / RNA polymerase II activity / RNA polymerase I complex / RNA polymerase III complex / RNA polymerase II, core complex / DNA-directed RNA polymerase complex / DNA-directed 5'-3' RNA polymerase activity / nucleic acid binding / protein dimerization activity / DNA-templated transcription / nucleolus / DNA binding / zinc ion binding
Similarity search - Function
Pol II subunit B9, C-terminal zinc ribbon / RNA polymerase RBP11 / 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. ...Pol II subunit B9, C-terminal zinc ribbon / RNA polymerase RBP11 / 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 / DNA-directed RNA polymerase, subunit RPB6 / DNA directed RNA polymerase, 7 kDa subunit / RNA polymerase archaeal subunit P/eukaryotic subunit RPABC4 / RNA polymerase subunit CX / DNA-directed RNA polymerase Rpb11, 13-16kDa subunit, conserved site / RNA polymerases L / 13 to 16 Kd subunits signature. / Zinc finger, TFIIS-type / Transcription factor S-II (TFIIS) / Zinc finger TFIIS-type profile. / C2C2 Zinc finger / 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 RPABC4/transcription elongation factor Spt4 / 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 / 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 / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / Uncharacterized protein / DNA-directed RNA polymerases I, II, and III subunit RPABC5 / Uncharacterized protein / DNA-directed RNA polymerase RpoA/D/Rpb3-type domain-containing protein / DNA-directed RNA polymerase RBP11-like dimerisation domain-containing protein / DNA-directed RNA polymerases II, IV and V subunit 8B / DNA-directed RNA polymerase subunit
Similarity search - Component
Biological speciesBrassica oleracea (wild cabbage)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsHu, H. / Xie, G. / Du, X. / Du, J.
Funding support1items
OrganizationGrant numberCountry
Other government
CitationJournal: Science / Year: 2023
Title: Structure and mechanism of the plant RNA polymerase V.
Authors: Guohui Xie / Xuan Du / Hongmiao Hu / Sisi Li / Xiaofeng Cao / Steven E Jacobsen / Jiamu Du /
Abstract: In addition to the conserved RNA polymerases I to III (Pols I to III) in eukaryotes, two atypical polymerases, Pols IV and V, specifically produce noncoding RNA in the RNA-directed DNA methylation ...In addition to the conserved RNA polymerases I to III (Pols I to III) in eukaryotes, two atypical polymerases, Pols IV and V, specifically produce noncoding RNA in the RNA-directed DNA methylation pathway in plants. Here, we report on the structures of cauliflower Pol V in the free and elongation conformations. A conserved tyrosine residue of NRPE2 stacks with a double-stranded DNA branch of the transcription bubble to potentially attenuate elongation by inducing transcription stalling. The nontemplate DNA strand is captured by NRPE2 to enhance backtracking, thereby increasing 3'-5' cleavage, which likely underpins Pol V's high fidelity. The structures also illuminate the mechanism of Pol V transcription stalling and enhanced backtracking, which may be important for Pol V's retention on chromatin to serve its function in tethering downstream factors for RNA-directed DNA methylation.
History
DepositionNov 21, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 22, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 5, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
T: DNA (34-MER)
N: DNA (34-MER)
P: RNA (5'-R(*UP*AP*UP*AP*UP*GP*CP*AP*UP*AP*AP*AP*GP*AP*CP*CP*AP*GP*GP*C)-3')
A: DNA-directed RNA polymerase V largest subunit
C: RPOLD domain-containing protein
F: DNA-directed RNA polymerases I, II, and III subunit RPABC2
J: DNA-directed RNA polymerases I, II, and III subunit RPABC5
K: RNA_pol_L_2 domain-containing protein
L: DNA-directed RNA polymerases II, IV and V subunit 12
H: DNA-directed RNA polymerases I, II, and III subunit RPABC3
I: DNA-directed RNA polymerase subunit
E: DNA-directed RNA polymerases I, II, and III subunit RPABC1
B: DNA-directed RNA polymerase IV and V subunit 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)518,69920
Polymers518,28213
Non-polymers4177
Water0
1


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

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Components

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DNA chain , 2 types, 2 molecules TN

#1: DNA chain DNA (34-MER)


Mass: 10482.763 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Brassica oleracea (wild cabbage)
#2: DNA chain DNA (34-MER)


Mass: 10463.749 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Brassica oleracea (wild cabbage)

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RNA chain , 1 types, 1 molecules P

#3: RNA chain RNA (5'-R(*UP*AP*UP*AP*UP*GP*CP*AP*UP*AP*AP*AP*GP*AP*CP*CP*AP*GP*GP*C)-3')


Mass: 6414.902 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Brassica oleracea (wild cabbage)

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DNA-directed RNA polymerase ... , 3 types, 3 molecules AIB

#4: Protein DNA-directed RNA polymerase V largest subunit


Mass: 222876.891 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Brassica oleracea (wild cabbage) / Tissue: inflorescence
#11: Protein DNA-directed RNA polymerase subunit / Polymerase


Mass: 13123.780 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Brassica oleracea (wild cabbage) / Tissue: inflorescence / References: UniProt: A0A3P6GD79
#13: Protein DNA-directed RNA polymerase IV and V subunit 2


Mass: 132377.766 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Brassica oleracea (wild cabbage) / Tissue: inflorescence

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Protein , 3 types, 3 molecules CKL

#5: Protein RPOLD domain-containing protein


Mass: 35484.070 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Brassica oleracea (wild cabbage) / Tissue: inflorescence / References: UniProt: A0A0D3D418
#8: Protein RNA_pol_L_2 domain-containing protein


Mass: 13538.273 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Brassica oleracea (wild cabbage) / Tissue: inflorescence / References: UniProt: A0A0D3DS91
#9: Protein DNA-directed RNA polymerases II, IV and V subunit 12 / RNA polymerase


Mass: 5983.796 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Brassica oleracea (wild cabbage) / Tissue: inflorescence / References: UniProt: A0A0D2ZPP3

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DNA-directed RNA polymerases I, II, and III subunit ... , 4 types, 4 molecules FJHE

#6: Protein DNA-directed RNA polymerases I, II, and III subunit RPABC2 / RNA polymerase


Mass: 16686.348 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Brassica oleracea (wild cabbage) / Tissue: inflorescence / References: UniProt: A0A0D3BZZ8
#7: Protein DNA-directed RNA polymerases I, II, and III subunit RPABC5 / RNA polymerase


Mass: 7982.328 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Brassica oleracea (wild cabbage) / Tissue: inflorescence / References: UniProt: A0A0D3AAT3
#10: Protein DNA-directed RNA polymerases I, II, and III subunit RPABC3 / RNA polymerase


Mass: 16607.275 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Brassica oleracea (wild cabbage) / Tissue: inflorescence / References: UniProt: A0A0D3DUD8
#12: Protein DNA-directed RNA polymerases I, II, and III subunit RPABC1 / RNA polymerase


Mass: 26260.105 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Brassica oleracea (wild cabbage) / Tissue: inflorescence

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Non-polymers , 2 types, 7 molecules

#14: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#15: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION

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Details

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

Component
IDNameTypeEntity IDParent-IDSource
1DNA-directed RNA polymerase V elongation complexCOMPLEX#1-#130MULTIPLE SOURCES
2DNA-directed RNA polymerasePolymeraseCOMPLEX#4-#131NATURAL
3DNA-RNACOMPLEX#1-#31SYNTHETIC
Source (natural)Organism: Brassica oleracea (wild cabbage) / Tissue: inflorescence
Buffer solutionpH: 7.8
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 280 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: OTHER / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 2.9975 sec. / Electron dose: 1.5625 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 5694

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Processing

EM software
IDNameVersionCategory
1RELION3.1.2particle selection
2EPU2.10.0.5RELimage acquisition
4RELION3.1.2CTF correction
7UCSF Chimera1.13.1model fitting
9PHENIX1.17.1model refinement
10RELION3.1.2initial Euler assignment
11RELION3.1.2final Euler assignment
13RELION3.1.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2750896
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 503124 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL

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