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- PDB-7um0: Structure of the phage AR9 non-virion RNA polymerase holoenzyme i... -

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Basic information

Entry
Database: PDB / ID: 7um0
TitleStructure of the phage AR9 non-virion RNA polymerase holoenzyme in complex with two DNA oligonucleotides containing the AR9 P077 promoter as determined by cryo-EM
Components
  • (DNA-directed RNA ...) x 5
  • DNA (5'-D(P*GP*UP*U)-3')
KeywordsTRANSCRIPTION / RNAP / deoxyuridine / template-strand promoter / sigma-like factor / gp226
Function / homology
Function and homology information


DNA-directed RNA polymerase complex / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / DNA-templated transcription / DNA binding
Similarity search - Function
DNA-directed RNA polymerase, subunit beta-prime / 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 / 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-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)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 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
Revision 2.0May 29, 2024Group: Advisory / Atomic model ...Advisory / Atomic model / Author supporting evidence / Data collection / Database references / Derived calculations / Polymer sequence / Source and taxonomy / Structure summary
Category: atom_site / atom_site_anisotrop ...atom_site / atom_site_anisotrop / em_entity_assembly / entity / entity_poly / entity_poly_seq / pdbx_entity_instance_feature / pdbx_entity_nonpoly / pdbx_entity_src_syn / pdbx_nonpoly_scheme / pdbx_poly_seq_scheme / pdbx_struct_assembly / pdbx_struct_assembly_gen / pdbx_struct_conn_angle / pdbx_unobs_or_zero_occ_atoms / pdbx_validate_close_contact / struct_asym / struct_conn / struct_ref / struct_ref_seq
Item: _atom_site.Cartn_x / _atom_site.Cartn_y ..._atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.group_PDB / _atom_site.label_asym_id / _atom_site.label_entity_id / _atom_site.label_seq_id / _atom_site_anisotrop.pdbx_auth_asym_id / _atom_site_anisotrop.pdbx_label_asym_id / _atom_site_anisotrop.pdbx_label_seq_id / _em_entity_assembly.entity_id_list / _pdbx_struct_assembly.oligomeric_count / _pdbx_struct_assembly.oligomeric_details / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _struct_conn.ptnr2_label_asym_id

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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
B: DNA (5'-D(P*GP*UP*U)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)315,9877
Polymers315,9226
Non-polymers651
Water0
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 / Polymerase


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
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 / Variant: Star / 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 / 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 / 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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DNA chain / Non-polymers , 2 types, 2 molecules B

#6: DNA chain DNA (5'-D(P*GP*UP*U)-3')


Mass: 864.582 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#7: 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 interestY

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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: AR9 nvRNAP promoter complex / Type: COMPLEX
Details: The complex consists of the AR9 nvRNAP holoenzyme and two copies of the same oligonucleotide containing the P077 promoter. Only three bases in one of the oligonucleotides are sufficiently ...Details: The complex consists of the AR9 nvRNAP holoenzyme and two copies of the same oligonucleotide containing the P077 promoter. Only three bases in one of the oligonucleotides are sufficiently ordered for atomic model building.
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.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / 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 FIELDBright-field microscopy / Nominal defocus max: 4000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 44 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k 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: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 106867 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
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: 88.16 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002122157
ELECTRON MICROSCOPYf_angle_d0.447729882
ELECTRON MICROSCOPYf_chiral_restr0.03893327
ELECTRON MICROSCOPYf_plane_restr0.00273830
ELECTRON MICROSCOPYf_dihedral_angle_d12.90422906

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