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- PDB-6rfl: Structure of the complete Vaccinia DNA-dependent RNA polymerase c... -

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Basic information

Entry
Database: PDB / ID: 6rfl
TitleStructure of the complete Vaccinia DNA-dependent RNA polymerase complex
Components
  • (DNA-dependent RNA polymerase subunit ...RNA polymerase) x 4
  • (DNA-directed RNA polymerase ...Polymerase) x 4
  • Large subunit of mRNA capping enzyme
  • Nucleoside triphosphate phosphohydrolase-I
  • Putative H4L RNA polymerase-associated transcription factor RAP94
  • Small subunit of mRNA capping enzyme
  • Transcription factor VETF 82kDa large subunit
  • Virion core protein
  • chr17.trna16-GlnTTG
KeywordsVIRAL PROTEIN / Vaccinia / RNA polymerase / RNA Polymerase complex / RNAP / vRNAP / complete vRNAP
Function / homology
Function and homology information


polynucleotide 5'-phosphatase / polynucleotide 5'-phosphatase activity / virion component => GO:0044423 / viral transcription / nucleotidyltransferase activity / ribonucleoside triphosphate phosphatase activity / DNA-templated transcription termination / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase ...polynucleotide 5'-phosphatase / polynucleotide 5'-phosphatase activity / virion component => GO:0044423 / viral transcription / nucleotidyltransferase activity / ribonucleoside triphosphate phosphatase activity / DNA-templated transcription termination / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / nucleoside-triphosphate phosphatase / mRNA guanylyltransferase activity / mRNA guanylyltransferase / mRNA (guanine-N7)-methyltransferase / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / DNA-binding transcription factor activity / DNA-templated transcription / positive regulation of DNA-templated transcription / DNA binding / zinc ion binding / ATP binding
Similarity search - Function
Virion core protein, vaccinia E11L type / Chordopoxvirus E11 protein / Poxvirus mRNA capping enzyme, small subunit / Poxvirus early transcription factor, large subunit / Nucleoside triphosphatase I, C-terminal / Poxvirus early transcription factor (VETF), large subunit / Nucleoside triphosphatase I C-terminal / mRNA capping enzyme, large subunit, ATPase/guanylyltransferase, virus / mRNA capping enzyme catalytic subunit, RNA triphosphatase domain / Poxvirus mRNA capping enzyme, small subunit ...Virion core protein, vaccinia E11L type / Chordopoxvirus E11 protein / Poxvirus mRNA capping enzyme, small subunit / Poxvirus early transcription factor, large subunit / Nucleoside triphosphatase I, C-terminal / Poxvirus early transcription factor (VETF), large subunit / Nucleoside triphosphatase I C-terminal / mRNA capping enzyme, large subunit, ATPase/guanylyltransferase, virus / mRNA capping enzyme catalytic subunit, RNA triphosphatase domain / Poxvirus mRNA capping enzyme, small subunit / Poxvirus mRNA capping enzyme, small subunit superfamily / Poxvirus mRNA capping enzyme, small subunit / RNA polymerase-associated transcription specificity factor Rap94 / RNA polymerase-associated transcription specificity factor, Rap94 / DNA-directed RNA polymerase, 18kDa subunit, poxviral / DNA-directed RNA polymerase, 35kDa subunit, poxviral / RNA polymerase, 22kDa subunit, poxviral / DNA-directed RNA polymerase, 19kDa subunit, poxviral / DNA-directed RNA polymerase, 7kDa polypeptide, chordopoxviral / RNA polymerase, 30kDa subunit, chordopox-type / RNA polymerase, 132kDa subunit, poxvirus-type / RNA polymerase, 30kDa subunit, chordopox-type, N-terminal / Poxvirus DNA-directed RNA polymerase, 18 kD subunit / Poxvirus DNA-directed RNA polymerase, 35 kD subunit / Poxvirus RNA polymerase 22 kDa subunit / Poxvirus DNA-directed RNA polymerase 19 kDa subunit / Chordopoxvirus DNA-directed RNA polymerase 7 kDa polypeptide (RPO7) / Poxvirus DNA dependent RNA polymerase 30kDa subunit / Poxvirus DNA dependent RNA polymerase / mRNA (guanine-N(7))-methyltransferase domain / mRNA cap guanine-N7 methyltransferase / mRNA (guanine-N(7))-methyltransferase domain / mRNA (guanine-N(7)-)-methyltransferase (EC 2.1.1.56) domain profile. / SNF2, N-terminal / SNF2-related domain / Zinc finger TFIIS-type signature. / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 5 / Zinc finger, TFIIS-type / Transcription factor S-II (TFIIS) / Zinc finger TFIIS-type profile. / C2C2 Zinc finger / Helicase conserved C-terminal domain / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 4 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 5 / 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 / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / S-adenosyl-L-methionine-dependent methyltransferase superfamily / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
RNA / RNA (> 10) / DNA-directed RNA polymerase 30 kDa polypeptide / Virion core protein / DNA-directed RNA polymerase 7 kDa subunit / DNA-directed RNA polymerase subunit / DNA-directed RNA polymerase 147 kDa polypeptide / Protein H4 / GTP--RNA guanylyltransferase / DNA-directed RNA polymerase 18 kDa subunit ...RNA / RNA (> 10) / DNA-directed RNA polymerase 30 kDa polypeptide / Virion core protein / DNA-directed RNA polymerase 7 kDa subunit / DNA-directed RNA polymerase subunit / DNA-directed RNA polymerase 147 kDa polypeptide / Protein H4 / GTP--RNA guanylyltransferase / DNA-directed RNA polymerase 18 kDa subunit / Nucleoside triphosphatase I / Virus termination factor small subunit / DNA-directed RNA polymerase 19 kDa subunit / ETF large subunit / DNA-directed RNA polymerase / DNA-directed RNA polymerase 35 kDa subunit / RNA polymerase-associated transcription-specificity factor RAP94
Similarity search - Component
Biological speciesVaccinia virus GLV-1h68
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.76 Å
AuthorsGrimm, C. / Hillen, S.H. / Bedenk, K. / Bartuli, J. / Neyer, S. / Zhang, Q. / Huettenhofer, A. / Erlacher, M. / Dienemann, C. / Schlosser, A. ...Grimm, C. / Hillen, S.H. / Bedenk, K. / Bartuli, J. / Neyer, S. / Zhang, Q. / Huettenhofer, A. / Erlacher, M. / Dienemann, C. / Schlosser, A. / Urlaub, H. / Boettcher, B. / Szalay, A.A. / Cramer, P. / Fischer, U.
Funding support Germany, 2items
OrganizationGrant numberCountry
German Research FoundationFi 573 7-2 Germany
German Research FoundationFi 573 18-1 Germany
CitationJournal: Cell / Year: 2019
Title: Structural Basis of Poxvirus Transcription: Vaccinia RNA Polymerase Complexes.
Authors: Clemens Grimm / Hauke S Hillen / Kristina Bedenk / Julia Bartuli / Simon Neyer / Qian Zhang / Alexander Hüttenhofer / Matthias Erlacher / Christian Dienemann / Andreas Schlosser / Henning ...Authors: Clemens Grimm / Hauke S Hillen / Kristina Bedenk / Julia Bartuli / Simon Neyer / Qian Zhang / Alexander Hüttenhofer / Matthias Erlacher / Christian Dienemann / Andreas Schlosser / Henning Urlaub / Bettina Böttcher / Aladar A Szalay / Patrick Cramer / Utz Fischer /
Abstract: Poxviruses encode a multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the host cytoplasm. We report cryo-EM structures of core and complete vRNAP enzymes ...Poxviruses encode a multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the host cytoplasm. We report cryo-EM structures of core and complete vRNAP enzymes from Vaccinia virus at 2.8 Å resolution. The vRNAP core enzyme resembles eukaryotic RNA polymerase II (Pol II) but also reveals many virus-specific features, including the transcription factor Rap94. The complete enzyme additionally contains the transcription factor VETF, the mRNA processing factors VTF/CE and NPH-I, the viral core protein E11, and host tRNA. This complex can carry out the entire early transcription cycle. The structures show that Rap94 partially resembles the Pol II initiation factor TFIIB, that the vRNAP subunit Rpo30 resembles the Pol II elongation factor TFIIS, and that NPH-I resembles chromatin remodeling enzymes. Together with the accompanying paper (Hillen et al., 2019), these results provide the basis for unraveling the mechanisms of poxvirus transcription and RNA processing.
History
DepositionApr 15, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 11, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 25, 2019Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

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Assembly

Deposited unit
B: DNA-dependent RNA polymerase subunit rpo132
E: DNA-directed RNA polymerase subunit
F: DNA-directed RNA polymerase 19 kDa subunit
G: DNA-dependent RNA polymerase subunit rpo18
I: Putative H4L RNA polymerase-associated transcription factor RAP94
J: DNA-dependent RNA polymerase subunit rpo7
L: Small subunit of mRNA capping enzyme
O: Large subunit of mRNA capping enzyme
R: Virion core protein
K: Transcription factor VETF 82kDa large subunit
U: chr17.trna16-GlnTTG
A: DNA-dependent RNA polymerase subunit rpo147
Q: Virion core protein
Y: Nucleoside triphosphate phosphohydrolase-I
C: DNA-directed RNA polymerase 35 kDa subunit
S: DNA-directed RNA polymerase 30 kDa polypeptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)843,67021
Polymers843,38416
Non-polymers2865
Water1,09961
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area110370 Å2
ΔGint-559 kcal/mol
Surface area242530 Å2
MethodPISA

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Components

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DNA-dependent RNA polymerase subunit ... , 4 types, 4 molecules BGJA

#1: Protein DNA-dependent RNA polymerase subunit rpo132 / RNA polymerase


Mass: 133526.859 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68 / Gene: GL194 / Production host: Vaccinia virus GLV-1h68 / References: UniProt: B9U1Q1, DNA-directed RNA polymerase
#4: Protein DNA-dependent RNA polymerase subunit rpo18 / RNA polymerase


Mass: 17917.195 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68 / Gene: GL147 / Production host: Vaccinia virus GLV-1h68 / References: UniProt: B9U1K4, DNA-directed RNA polymerase
#6: Protein DNA-dependent RNA polymerase subunit rpo7 / RNA polymerase


Mass: 7299.715 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68 / Gene: GL107 / Production host: Vaccinia virus GLV-1h68 / References: UniProt: B9U1G3, DNA-directed RNA polymerase
#12: Protein DNA-dependent RNA polymerase subunit rpo147 / RNA polymerase


Mass: 146995.703 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68 / Gene: GL125 / Production host: Vaccinia virus GLV-1h68 / References: UniProt: B9U1I2, DNA-directed RNA polymerase

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DNA-directed RNA polymerase ... , 4 types, 4 molecules EFCS

#2: Protein DNA-directed RNA polymerase subunit / Polymerase


Mass: 21365.740 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68 / Gene: GL123 / Production host: Vaccinia virus GLV-1h68 / References: UniProt: B9U1I0, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase 19 kDa subunit


Mass: 19020.088 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68 / Gene: GL167 / Production host: Vaccinia virus GLV-1h68 / References: UniProt: B9U1M4, DNA-directed RNA polymerase
#14: Protein DNA-directed RNA polymerase 35 kDa subunit


Mass: 35430.676 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68 / Gene: GL205 / Production host: Vaccinia virus GLV-1h68 / References: UniProt: B9U1R2, DNA-directed RNA polymerase
#15: Protein DNA-directed RNA polymerase 30 kDa polypeptide


Mass: 30074.293 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68 / Gene: GL076 / Production host: Vaccinia virus GLV-1h68 / References: UniProt: B9U1D1, DNA-directed RNA polymerase

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Protein , 6 types, 7 molecules ILORQKY

#5: Protein Putative H4L RNA polymerase-associated transcription factor RAP94 / RAP94 / RNA polymerase rpo94 / RNA polymerase-associated transcription-specificity factor RAP94 / VACV098


Mass: 93667.633 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68
Gene: H4L, List098, LIVPclone14_110, VAC_DPP10_113, VAC_DPP11_113, VAC_DPP12_113, VAC_DPP13_113, VAC_DPP15_113, VAC_DPP16_113, VAC_DPP17_113, VAC_DPP19_113, VAC_DPP20_113, VAC_DPP21_113, VAC_DPP9_ ...Gene: H4L, List098, LIVPclone14_110, VAC_DPP10_113, VAC_DPP11_113, VAC_DPP12_113, VAC_DPP13_113, VAC_DPP15_113, VAC_DPP16_113, VAC_DPP17_113, VAC_DPP19_113, VAC_DPP20_113, VAC_DPP21_113, VAC_DPP9_113, VACAC2_113, VACCL3_113, VACV_098, VACV_TT9_123
Production host: Vaccinia virus GLV-1h68
References: UniProt: Q1PIU7, UniProt: B9U1I7*PLUS, DNA-directed RNA polymerase
#7: Protein Small subunit of mRNA capping enzyme


Mass: 33396.656 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68 / Gene: GL155 / Production host: Vaccinia virus GLV-1h68
References: UniProt: B9U1L2, mRNA (guanine-N7)-methyltransferase
#8: Protein Large subunit of mRNA capping enzyme


Mass: 96888.758 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68 / Gene: GL135 / Production host: Vaccinia virus GLV-1h68 / References: UniProt: B9U1J2
#9: Protein Virion core protein


Mass: 14914.090 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68 / Gene: GL087 / Production host: Vaccinia virus GLV-1h68 / References: UniProt: B9U1E2
#10: Protein Transcription factor VETF 82kDa large subunit


Mass: 82398.906 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68 / Gene: GL169 / Production host: Vaccinia virus GLV-1h68 / References: UniProt: B9U1M6
#13: Protein Nucleoside triphosphate phosphohydrolase-I


Mass: 72465.320 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68 / Gene: GL151 / Production host: Vaccinia virus GLV-1h68
References: UniProt: B9U1K8, nucleoside-triphosphate phosphatase

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RNA chain , 1 types, 1 molecules U

#11: RNA chain chr17.trna16-GlnTTG


Mass: 23108.650 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)

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Non-polymers , 3 types, 66 molecules

#16: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
#17: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#18: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 61 / Source method: isolated from a natural source / Formula: H2O

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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
1Complete Vaccinia DNA-dependent RNA polymerase complexCOMPLEX#1-#150MULTIPLE SOURCES
2Vaccinia DNA-dependent RNA polymerase complexCOMPLEX#1-#10, #12-#151RECOMBINANT
3RNACOMPLEX#111RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Vaccinia virus GLV-1H68502057
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Vaccinia virus GLV-1H68502057
23Homo sapiens (human)9606
Buffer solutionpH: 7.5
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 50 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.15.1_3469: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.76 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 618338 / Symmetry type: POINT

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