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
National Natural Science Foundation of China (NSFC)
31822001
China
Citation
Journal: 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
Deposition
May 15, 2021
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Header (metadata) release
Dec 29, 2021
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Map release
Dec 29, 2021
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Update
Jun 5, 2024
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Current status
Jun 5, 2024
Processing site: PDBj / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Name: RNA (39-MER) / type: rna / ID: 13 / Number of copies: 1
Source (natural)
Organism: synthetic construct (others)
Molecular weight
Theoretical: 12.21502 KDa
Sequence
String:
UUUUAUCGAG AGGUACUUUU CUUUUCUUUC UUCUUCGGU
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Macromolecule #15: RNA (5'-R(*CP*CP*GP*A)-3')
Macromolecule
Name: RNA (5'-R(*CP*CP*GP*A)-3') / type: rna / ID: 15 / Number of copies: 1
Source (natural)
Organism: synthetic construct (others)
Molecular weight
Theoretical: 1.239818 KDa
Sequence
String:
CCGA
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Macromolecule #16: ZINC ION
Macromolecule
Name: ZINC ION / type: ligand / ID: 16 / Number of copies: 8 / Formula: ZN
Molecular weight
Theoretical: 65.409 Da
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Macromolecule #17: MAGNESIUM ION
Macromolecule
Name: MAGNESIUM ION / type: ligand / ID: 17 / Number of copies: 2 / Formula: MG
Molecular weight
Theoretical: 24.305 Da
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Concentration
1.1 mg/mL
Buffer
pH: 7.5 Component:
Concentration
Formula
Name
20.0 mmol/L
C2H18N2O4S
HEPES
100.0 mmol/L
NaCl
Sodium chloride
5.0 mmol/L
MgCl2
magnesium chloride
2.0 mmol/L
C4H10O2S2
DL-dithiothreitol
Grid
Model: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 120 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: OTHER
Vitrification
Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 282.65 K / Instrument: FEI VITROBOT MARK III
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-32 / Average electron dose: 1.95 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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