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- PDB-7ap8: Atomic structure of the poxvirus initially transcribing complex i... -

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Basic information

Entry
Database: PDB / ID: 7ap8
TitleAtomic structure of the poxvirus initially transcribing complex in conformation 2
Components
  • (DNA-directed RNA polymerase ...) x 7
  • (Synthetic promoter DNA oligomer, ...) x 2
  • DNA-directed RNA polymerase
  • Protein H4
  • RNA (5'-R(P*AP*UP*AP*AP*A)-3')
KeywordsTRANSCRIPTION / Vaccinia / Virus / RNA polymerase / DNA-dependent / initiation complex / PIC / poxvirus / poxviridae
Function / homology
Function and homology information


viral transcription / RNA polymerase I activity / DNA-directed RNA polymerase complex / nucleotidyltransferase activity / DNA-templated transcription termination / virion component / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / DNA-binding transcription factor activity ...viral transcription / RNA polymerase I activity / DNA-directed RNA polymerase complex / nucleotidyltransferase activity / DNA-templated transcription termination / virion component / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / DNA-binding transcription factor activity / DNA-templated transcription / DNA binding / zinc ion binding / metal ion binding
Similarity search - Function
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 ...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 / 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 / RPB6/omega subunit-like superfamily / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / 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
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / DNA-directed RNA polymerase subunit / DNA-directed RNA polymerase 30 kDa polypeptide / RNA polymerase-associated transcription-specificity factor RAP94 / DNA-directed RNA polymerase 147 kDa polypeptide / DNA-directed RNA polymerase 35 kDa subunit / DNA-directed RNA polymerase / DNA-directed RNA polymerase 18 kDa subunit ...DNA / DNA (> 10) / RNA / DNA-directed RNA polymerase subunit / DNA-directed RNA polymerase 30 kDa polypeptide / RNA polymerase-associated transcription-specificity factor RAP94 / DNA-directed RNA polymerase 147 kDa polypeptide / DNA-directed RNA polymerase 35 kDa subunit / DNA-directed RNA polymerase / DNA-directed RNA polymerase 18 kDa subunit / DNA-directed RNA polymerase 19 kDa subunit / DNA-directed RNA polymerase 7 kDa subunit
Similarity search - Component
Biological speciesVaccinia virus GLV-1h68
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.15 Å
AuthorsGrimm, C. / Bartuli, J. / Fischer, U.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)Fi-573/20-1 Germany
CitationJournal: Nat Struct Mol Biol / Year: 2021
Title: Structural basis of the complete poxvirus transcription initiation process.
Authors: Clemens Grimm / Julia Bartuli / Bettina Boettcher / Aladar A Szalay / Utz Fischer /
Abstract: Poxviruses express their genes in the cytoplasm of infected cells using a virus-encoded multi-subunit polymerase (vRNAP) and unique transcription factors. We present cryo-EM structures that uncover ...Poxviruses express their genes in the cytoplasm of infected cells using a virus-encoded multi-subunit polymerase (vRNAP) and unique transcription factors. We present cryo-EM structures that uncover the complete transcription initiation phase of the poxvirus vaccinia. In the pre-initiation complex, the heterodimeric early transcription factor VETFs/l adopts an arc-like shape spanning the polymerase cleft and anchoring upstream and downstream promoter elements. VETFI emerges as a TBP-like protein that inserts asymmetrically into the DNA major groove, triggers DNA melting, ensures promoter recognition and enforces transcription directionality. The helicase VETFs fosters promoter melting and the phospho-peptide domain (PPD) of vRNAP subunit Rpo30 enables transcription initiation. An unprecedented upstream promoter scrunching mechanism assisted by the helicase NPH-I probably fosters promoter escape and transition into elongation. Our structures shed light on unique mechanisms of poxviral gene expression and aid the understanding of thus far unexplained universal principles in transcription.
History
DepositionOct 16, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 6, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 13, 2021Group: Data collection / Database references
Category: citation / database_PDB_rev ...citation / database_PDB_rev / database_PDB_rev_record / em_admin / pdbx_database_proc / pdbx_seq_map_depositor_info
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update / _pdbx_seq_map_depositor_info.one_letter_code_mod
Revision 1.2Oct 20, 2021Group: Data collection / Database references
Category: citation / em_admin ...citation / em_admin / pdbx_database_proc / pdbx_seq_map_depositor_info
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update / _pdbx_seq_map_depositor_info.one_letter_code_mod

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Structure visualization

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Assembly

Deposited unit
A: DNA-directed RNA polymerase 147 kDa polypeptide
B: DNA-directed RNA polymerase
C: DNA-directed RNA polymerase 35 kDa subunit
E: DNA-directed RNA polymerase subunit
F: DNA-directed RNA polymerase 19 kDa subunit
G: DNA-directed RNA polymerase 18 kDa subunit
I: Protein H4
J: DNA-directed RNA polymerase 7 kDa subunit
N: Synthetic promoter DNA oligomer, non-template strand
P: RNA (5'-R(P*AP*UP*AP*AP*A)-3')
S: DNA-directed RNA polymerase 30 kDa polypeptide
T: Synthetic promoter DNA oligomer, template strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)543,88817
Polymers543,60212
Non-polymers2865
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area74550 Å2
ΔGint-390 kcal/mol
Surface area149850 Å2
MethodPISA

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Components

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DNA-directed RNA polymerase ... , 7 types, 7 molecules ACEFGJS

#1: Protein DNA-directed RNA polymerase 147 kDa polypeptide


Mass: 146995.703 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus GLV-1h68 / References: UniProt: Q1PIV1, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase 35 kDa subunit


Mass: 35430.676 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus GLV-1h68 / References: UniProt: Q49PG1, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit


Mass: 21365.740 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus GLV-1h68 / References: UniProt: A4GDF1, DNA-directed RNA polymerase
#5: Protein DNA-directed RNA polymerase 19 kDa subunit


Mass: 19020.088 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus GLV-1h68 / References: UniProt: Q49QC8, DNA-directed RNA polymerase
#6: Protein DNA-directed RNA polymerase 18 kDa subunit


Mass: 17917.195 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus GLV-1h68 / References: UniProt: Q49PL6, DNA-directed RNA polymerase
#8: Protein DNA-directed RNA polymerase 7 kDa subunit


Mass: 7299.715 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus GLV-1h68 / References: UniProt: Q49QI2, DNA-directed RNA polymerase
#11: Protein DNA-directed RNA polymerase 30 kDa polypeptide


Mass: 29834.359 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus GLV-1h68 / References: UniProt: H2DZ00, DNA-directed RNA polymerase

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Protein , 2 types, 2 molecules BI

#2: Protein DNA-directed RNA polymerase


Mass: 133526.859 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus GLV-1h68 / References: UniProt: Q49PH2, DNA-directed RNA polymerase
#7: Protein Protein H4 / RNA polymerase-associated transcription-specificity factor RAP94 / RPO-associated protein of 94 kDa


Mass: 93667.633 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Vaccinia virus GLV-1h68 / References: UniProt: Q1PIU7

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Synthetic promoter DNA oligomer, ... , 2 types, 2 molecules NT

#9: DNA chain Synthetic promoter DNA oligomer, non-template strand


Mass: 18417.867 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Vaccinia virus GLV-1h68
#12: DNA chain Synthetic promoter DNA oligomer, template strand


Mass: 18547.941 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Vaccinia virus GLV-1h68

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RNA chain , 1 types, 1 molecules P

#10: RNA chain RNA (5'-R(P*AP*UP*AP*AP*A)-3')


Mass: 1578.032 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vaccinia virus GLV-1h68 / Production host: Vaccinia virus GLV-1h68

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Non-polymers , 2 types, 5 molecules

#13: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#14: 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

Component
IDNameTypeEntity IDParent-IDSource
1Vaccinia transcription pre-initiation complexCOMPLEX#1-#120MULTIPLE SOURCES
2Vaccinia transcription pre-initiation complexCOMPLEX#1-#8, #111NATURAL
3RNACOMPLEX#101RECOMBINANT
4DNACOMPLEX#9, #121RECOMBINANT
Molecular weightValue: 0.67 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Vaccinia virus GLV-1h68502057
23Vaccinia virus GLV-1h68502057
34Vaccinia virus GLV-1h68502057
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
13Vaccinia virus GLV-1h68502057
24synthetic construct (others)32630
Buffer solutionpH: 7.5
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: monodisperse sample prepared by sucrose gradient centrifugation
Specimen supportGrid material: COPPER / Grid type: Quantifoil
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
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 74.99 sec. / Electron dose: 78.9 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 3

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 73035 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00332235
ELECTRON MICROSCOPYf_angle_d0.54443747
ELECTRON MICROSCOPYf_dihedral_angle_d18.46612195
ELECTRON MICROSCOPYf_chiral_restr0.0434952
ELECTRON MICROSCOPYf_plane_restr0.0035392

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