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- PDB-7um1: Structure of bacteriophage AR9 non-virion RNAP polymerase holoenz... -

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Basic information

Entry
Database: PDB / ID: 7um1
TitleStructure of bacteriophage AR9 non-virion RNAP polymerase holoenzyme determined by cryo-EM
Components(DNA-directed RNA ...) x 5
KeywordsTRANSCRIPTION / RNAP / deoxyuridine / template-strand promoter / sigma-like factor / gp226
Function / homology
Function and homology information


RNA polymerase II activity / DNA-directed RNA polymerase complex / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / DNA-templated transcription / DNA binding / metal ion binding
Similarity search - Function
DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 5 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / 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-directed RNA polymerase / DNA-directed RNA polymerase beta subunit / DNA-directed RNA polymerase / DNA-directed RNA polymerase subunit / DNA-directed RNA polymerase
Similarity search - Component
Biological speciesBacillus phage AR9 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsLeiman, P.G. / Fraser, A. / Sokolova, M.L.
Funding support United States, 1items
OrganizationGrant numberCountry
Other governmentUTMB internal United States
CitationJournal: Nat Commun / Year: 2022
Title: Structural basis of template strand deoxyuridine promoter recognition by a viral RNA polymerase.
Authors: Alec Fraser / Maria L Sokolova / Arina V Drobysheva / Julia V Gordeeva / Sergei Borukhov / John Jumper / Konstantin V Severinov / Petr G Leiman /
Abstract: Recognition of promoters in bacterial RNA polymerases (RNAPs) is controlled by sigma subunits. The key sequence motif recognized by the sigma, the -10 promoter element, is located in the non-template ...Recognition of promoters in bacterial RNA polymerases (RNAPs) is controlled by sigma subunits. The key sequence motif recognized by the sigma, the -10 promoter element, is located in the non-template strand of the double-stranded DNA molecule ~10 nucleotides upstream of the transcription start site. Here, we explain the mechanism by which the phage AR9 non-virion RNAP (nvRNAP), a bacterial RNAP homolog, recognizes the -10 element of its deoxyuridine-containing promoter in the template strand. The AR9 sigma-like subunit, the nvRNAP enzyme core, and the template strand together form two nucleotide base-accepting pockets whose shapes dictate the requirement for the conserved deoxyuridines. A single amino acid substitution in the AR9 sigma-like subunit allows one of these pockets to accept a thymine thus expanding the promoter consensus. Our work demonstrates the extent to which viruses can evolve host-derived multisubunit enzymes to make transcription of their own genes independent of the host.
History
DepositionApr 5, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 6, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 14, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA-directed RNA polymerase subunit
d: DNA-directed RNA polymerase beta' subunit
c: DNA-directed RNA polymerase beta subunit
D: DNA-directed RNA polymerase
C: DNA-directed RNA polymerase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)315,1226
Polymers315,0575
Non-polymers651
Water00
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-directed RNA ... , 5 types, 5 molecules AdcDC

#1: Protein DNA-directed RNA polymerase subunit


Mass: 54917.449 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Promoter-specificity subunit / Source: (gene. exp.) Bacillus phage AR9 (virus) / Gene: AR9_g226 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): Star / References: UniProt: A0A172JIC8
#2: Protein DNA-directed RNA polymerase beta' subunit


Mass: 51947.844 Da / Num. of mol.: 1 / Mutation: N-terminal His-tag
Source method: isolated from a genetically manipulated source
Details: N-terminal part of beta-prime subunit / Source: (gene. exp.) Bacillus phage AR9 (virus) / Gene: AR9_g270 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): Star / References: UniProt: A0A172JIH0
#3: Protein DNA-directed RNA polymerase beta subunit


Mass: 58112.289 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N-terminal part of beta subunit / Source: (gene. exp.) Bacillus phage AR9 (virus) / Gene: AR9_g105 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): Star / References: UniProt: A0A172JI16
#4: Protein DNA-directed RNA polymerase


Mass: 73100.977 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: C-terminal part of beta-prime subunit / Source: (gene. exp.) Bacillus phage AR9 (virus) / Gene: AR9_g154 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): Star
References: UniProt: A0A172JI62, DNA-directed RNA polymerase
#5: Protein DNA-directed RNA polymerase


Mass: 76978.383 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: C-terminal part of beta subunit / Source: (gene. exp.) Bacillus phage AR9 (virus) / Gene: AR9_g089 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): Star
References: UniProt: A0A172JHZ2, DNA-directed RNA polymerase

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Non-polymers , 1 types, 1 molecules

#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / 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: Bacteriophage AR9 non-virion RNA polymerase holoenzyme
Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Bacillus phage AR9 (virus)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3) Star
Buffer solutionpH: 6.8
SpecimenConc.: 20 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 43.2 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameCategory
7Cootmodel fitting
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 104471 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Atomic model buildingPDB-ID: 7S01

7s01


Accession code: 7S01 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 120.22 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004918684
ELECTRON MICROSCOPYf_angle_d0.795925187
ELECTRON MICROSCOPYf_chiral_restr0.04572804
ELECTRON MICROSCOPYf_plane_restr0.00513241
ELECTRON MICROSCOPYf_dihedral_angle_d4.95272429

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