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- PDB-8hyj: A cryo-EM structure of KTF1-bound polymerase V transcription elon... -

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Entry
Database: PDB / ID: 8hyj
TitleA cryo-EM structure of KTF1-bound polymerase V transcription elongation complex
Components
  • (DNA (48-MER)) x 2
  • (DNA-directed RNA polymerase V subunit ...) x 3
  • (DNA-directed RNA polymerases II, IV and V subunit ...) x 5
  • (DNA-directed RNA polymerases IV and V subunit ...) x 3
  • DNA-directed RNA polymerases II and V subunit 8A
  • Protein RNA-directed DNA methylation 3
  • RNA (30-MER)
KeywordsTRANSCRIPTION / Pol V
Function / homology
Function and homology information


RNA polymerase IV complex / transposable element silencing by siRNA-mediated heterochromatin formation / RNA polymerase V complex / gene silencing by siRNA-directed DNA methylation / DNA-templated transcription elongation / regulatory ncRNA-mediated post-transcriptional gene silencing / siRNA processing / regulatory ncRNA-mediated gene silencing / plastid / RNA polymerase complex ...RNA polymerase IV complex / transposable element silencing by siRNA-mediated heterochromatin formation / RNA polymerase V complex / gene silencing by siRNA-directed DNA methylation / DNA-templated transcription elongation / regulatory ncRNA-mediated post-transcriptional gene silencing / siRNA processing / regulatory ncRNA-mediated gene silencing / plastid / RNA polymerase complex / regulation of immune response / defense response to fungus / heterochromatin / RNA polymerase II, core complex / regulation of DNA-templated transcription elongation / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / heterochromatin formation / nucleic acid binding / transcription by RNA polymerase II / protein dimerization activity / nuclear body / nucleotide binding / DNA-templated transcription / chromatin binding / regulation of transcription by RNA polymerase II / nucleolus / mitochondrion / DNA binding / RNA binding / zinc ion binding / nucleoplasm / metal ion binding / nucleus
Similarity search - Function
RDM3 beta-solenoid / DNA-directed RNA polymerase IV/V subunit 1, C-terminal / DNA-directed RNA polymerase IV/V subunit 1, N-terminal / Protein of unknown function (DUF3223) / Transcription elongation factor SPT5, KOWx domain / Transcription elongation factor SPT5, KOW1 domain / Transcription elongation factor SPT5, fourth KOW domain / NGN domain, eukaryotic / Spt5, KOW domain repeat 1 / Spt5, KOW domain repeat 4 ...RDM3 beta-solenoid / DNA-directed RNA polymerase IV/V subunit 1, C-terminal / DNA-directed RNA polymerase IV/V subunit 1, N-terminal / Protein of unknown function (DUF3223) / Transcription elongation factor SPT5, KOWx domain / Transcription elongation factor SPT5, KOW1 domain / Transcription elongation factor SPT5, fourth KOW domain / NGN domain, eukaryotic / Spt5, KOW domain repeat 1 / Spt5, KOW domain repeat 4 / NGN domain / Transcription elongation factor SPT5 / Early transcription elongation factor of RNA pol II, NGN section / NusG, N-terminal domain superfamily / DNA-directed RNA polymerase II subunit Rpb4-like / RNA polymerase Rpb4/RPC9, core / DNA-directed RNA-polymerase II subunit / Pol II subunit B9, C-terminal zinc ribbon / RNA polymerase RBP11 / Rpb4/RPC9 superfamily / RNA polymerase subunit Rpb4/RPC9 / RNA polymerase Rpb4 / RNA polymerase Rpb7-like , N-terminal / RNA polymerase Rpb7-like, N-terminal domain superfamily / RNA polymerase subunit Rpb7-like / SHS2 domain found in N terminus of Rpb7p/Rpc25p/MJ0397 / HRDC-like superfamily / 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 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 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, 7 kDa subunit / RNA polymerase archaeal subunit P/eukaryotic subunit RPABC4 / RNA polymerase subunit CX / 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 Rpb11, 13-16kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo11 / RNA polymerases L / 13 to 16 Kd subunits signature. / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / Zinc finger, TFIIS-type / Transcription factor S-II (TFIIS) / Zinc finger TFIIS-type profile. / C2C2 Zinc finger / DNA-directed RNA polymerase, RBP11-like dimerisation domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase, subunit H/Rpb5 C-terminal / DNA-directed RNA polymerase subunit Rpo5/Rpb5 / RPB5-like RNA polymerase subunit superfamily / RNA polymerase Rpb5, C-terminal domain / 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. / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 superfamily / 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 / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, N-terminal / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase I subunit A N-terminus / RNA polymerase, RBP11-like subunit / RNA polymerase, beta subunit, conserved site
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / DNA-directed RNA polymerase V subunit 7 / Protein RNA-directed DNA methylation 3 / DNA-directed RNA polymerases II and V subunit 8A / DNA-directed RNA polymerases II, IV and V subunit 11 / DNA-directed RNA polymerases IV and V subunit 3B / DNA-directed RNA polymerase V subunit 1 ...DNA / DNA (> 10) / RNA / RNA (> 10) / DNA-directed RNA polymerase V subunit 7 / Protein RNA-directed DNA methylation 3 / DNA-directed RNA polymerases II and V subunit 8A / DNA-directed RNA polymerases II, IV and V subunit 11 / DNA-directed RNA polymerases IV and V subunit 3B / DNA-directed RNA polymerase V subunit 1 / DNA-directed RNA polymerases IV and V subunit 4 / 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 polymerase V subunit 5A
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsZhang, H. / Zhang, Y.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)2020YFA0907902 China
National Natural Science Foundation of China (NSFC)JCYJ-SHFY-2022-012 China
CitationJournal: Nat Commun / Year: 2023
Title: A cryo-EM structure of KTF1-bound polymerase V transcription elongation complex.
Authors: Hong-Wei Zhang / Kun Huang / Zhan-Xi Gu / Xiao-Xian Wu / Jia-Wei Wang / Yu Zhang /
Abstract: De novo DNA methylation in plants relies on transcription of RNA polymerase V (Pol V) along with KTF1, which produce long non-coding RNAs for recruitment and assembly of the DNA methylation machinery. ...De novo DNA methylation in plants relies on transcription of RNA polymerase V (Pol V) along with KTF1, which produce long non-coding RNAs for recruitment and assembly of the DNA methylation machinery. Here, we report a cryo-EM structure of the Pol V transcription elongation complex bound to KTF1. The structure reveals the conformation of the structural motifs in the active site of Pol V that accounts for its inferior RNA-extension ability. The structure also reveals structural features of Pol V that prevent it from interacting with the transcription factors of Pol II and Pol IV. The KOW5 domain of KTF1 binds near the RNA exit channel of Pol V providing a scaffold for the proposed recruitment of Argonaute proteins to initiate the assembly of the DNA methylation machinery. The structure provides insight into the Pol V transcription elongation process and the role of KTF1 during Pol V transcription-coupled DNA methylation.
History
DepositionJan 6, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jun 21, 2023Provider: repository / Type: Initial release
Revision 1.1May 29, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: DNA-directed RNA polymerase V subunit 1
B: DNA-directed RNA polymerases IV and V subunit 2
C: DNA-directed RNA polymerases IV and V subunit 3B
D: DNA-directed RNA polymerases IV and V subunit 4
E: DNA-directed RNA polymerase V subunit 5A
F: DNA-directed RNA polymerases II, IV and V subunit 6A
G: DNA-directed RNA polymerase V subunit 7
H: DNA-directed RNA polymerases II and V subunit 8A
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
N: DNA (48-MER)
P: RNA (30-MER)
T: DNA (48-MER)
W: Protein RNA-directed DNA methylation 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)727,23821
Polymers726,95216
Non-polymers2865
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA-directed RNA polymerase V subunit ... , 3 types, 3 molecules AEG

#1: Protein DNA-directed RNA polymerase V subunit 1 / DNA-directed RNA polymerase D subunit 1b / AtNRPD1b / Nuclear RNA polymerase D 1b / DNA-directed ...DNA-directed RNA polymerase D subunit 1b / AtNRPD1b / Nuclear RNA polymerase D 1b / DNA-directed RNA polymerase E subunit 1 / Nuclear RNA polymerase E 1 / Protein DEFECTIVE IN MERISTEM SILENCING 5 / Protein DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 3 / Protein RNA-DIRECTED DNA METHYLATION DEFECTIVE 1 / RNA polymerase IV subunit 1 / POL IV 1


Mass: 218495.594 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress)
Gene: NRPE1, DMS5, DRD3, NRPD1b, RMD1, RPE1, At2g40030, T28M21.19
Production host: Arabidopsis thaliana (thale cress) / References: UniProt: Q5D869, DNA-directed RNA polymerase
#5: Protein DNA-directed RNA polymerase V subunit 5A


Mass: 25626.555 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Arabidopsis thaliana (thale cress) / References: UniProt: Q9M1J2
#7: Protein DNA-directed RNA polymerase V subunit 7


Mass: 20287.941 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Arabidopsis thaliana (thale cress) / References: UniProt: A6QRA1

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DNA-directed RNA polymerases IV and V subunit ... , 3 types, 3 molecules BCD

#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 IV and V subunit 3B / DNA-directed RNA polymerase II 36 kDa polypeptide B / DNA-directed RNA polymerase II subunit RPB3-B ...DNA-directed RNA polymerase II 36 kDa polypeptide B / DNA-directed RNA polymerase II subunit RPB3-B / RNA polymerase II subunit 3-B / RNA polymerase II subunit B3-B


Mass: 35617.375 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Arabidopsis thaliana (thale cress) / References: UniProt: Q39212
#4: Protein DNA-directed RNA polymerases IV and V subunit 4 / Protein RNA-DIRECTED DNA METHYLATION 2 / RNA polymerase II / Rpb4 / core protein


Mass: 22447.604 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Arabidopsis thaliana (thale cress) / References: UniProt: Q6DBA5

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DNA-directed RNA polymerases II, IV and V subunit ... , 5 types, 5 molecules FIJKL

#6: Protein DNA-directed RNA polymerases II, IV and V subunit 6A / 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
#9: Protein DNA-directed RNA polymerases II, IV and V subunit 9A


Mass: 13297.145 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Arabidopsis thaliana (thale cress) / References: UniProt: Q6NLH0
#10: Protein DNA-directed RNA polymerases II, IV and V subunit 10 / 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
#11: Protein DNA-directed RNA polymerases II, IV and V subunit 11 / 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
#12: Protein DNA-directed RNA polymerases II, IV and V subunit 12 / 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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Protein , 2 types, 2 molecules HW

#8: Protein DNA-directed RNA polymerases II and V subunit 8A / RNA polymerase I / II and III 16.5 kDa subunit / AtRPABC16.5


Mass: 16533.148 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Arabidopsis thaliana (thale cress) / References: UniProt: O81097
#16: Protein Protein RNA-directed DNA methylation 3 / KOW domain-containing transcription factor 1 / Protein SPT5-like


Mass: 158254.062 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: RDM3, KTF1, SPT5L, At5g04290, T19N18.20 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: F4JW79

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DNA chain , 2 types, 2 molecules NT

#13: DNA chain DNA (48-MER)


Mass: 14926.555 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#15: DNA chain DNA (48-MER)


Mass: 14643.407 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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RNA chain , 1 types, 1 molecules P

#14: RNA chain RNA (30-MER)


Mass: 9492.561 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 5 molecules

#17: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#18: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn

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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: KTF1-bound polymerase V transcription elongation complex
Type: COMPLEX / Entity ID: #1-#16 / Source: MULTIPLE SOURCES
Molecular weightValue: 608.5 kDa/nm / Experimental value: NO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Buffer solutionpH: 7.4
SpecimenConc.: 2.4 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

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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 FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.16_3549: / Category: model refinement
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30359 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 161.95 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01529256
ELECTRON MICROSCOPYf_angle_d1.479139951
ELECTRON MICROSCOPYf_chiral_restr0.16714636
ELECTRON MICROSCOPYf_plane_restr0.00724805
ELECTRON MICROSCOPYf_dihedral_angle_d18.666517374

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