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- PDB-6zca: Structure of the B. subtilis RNA POLYMERASE in complex with HelD ... -

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Basic information

Entry
Database: PDB / ID: 6zca
TitleStructure of the B. subtilis RNA POLYMERASE in complex with HelD (monomer)
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 3
  • DNA helicaseHelicase
  • Probable DNA-directed RNA polymerase subunit delta,Probable DNA-directed RNA polymerase subunit delta,Probable DNA-directed RNA polymerase subunit delta
  • RNA polymerase subunit omega
KeywordsTRANSCRIPTION / TRANSCRIPTION/DNA/RNA / DNA-DEPENDENT RNA POLYMERASE / BACTERIAL / Helicase
Function / homology
Function and homology information


nucleoid / recombinational repair / 3'-5' DNA helicase activity / DNA helicase activity / DNA-directed RNA polymerase complex / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / DNA helicase / protein dimerization activity ...nucleoid / recombinational repair / 3'-5' DNA helicase activity / DNA helicase activity / DNA-directed RNA polymerase complex / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / DNA helicase / protein dimerization activity / hydrolase activity / response to antibiotic / DNA-templated transcription / regulation of DNA-templated transcription / magnesium ion binding / ATP hydrolysis activity / DNA binding / zinc ion binding / ATP binding / cytosol / cytoplasm
Similarity search - Function
: / DNA-directed RNA polymerase subunit delta / DNA-directed RNA polymerase subunit delta, N-terminal domain superfamily / RNA polymerase epsilon subunit / RNA polymerase epsilon subunit / ASXL, HARE-HTH domain / HB1, ASXL, restriction endonuclease HTH domain / HARE-type HTH domain profile. / AAA domain / UvrD-like helicase C-terminal domain ...: / DNA-directed RNA polymerase subunit delta / DNA-directed RNA polymerase subunit delta, N-terminal domain superfamily / RNA polymerase epsilon subunit / RNA polymerase epsilon subunit / ASXL, HARE-HTH domain / HB1, ASXL, restriction endonuclease HTH domain / HARE-type HTH domain profile. / AAA domain / UvrD-like helicase C-terminal domain / UvrD-like helicase C-terminal domain / UvrD/REP helicase N-terminal domain / UvrD-like helicase, ATP-binding domain / UvrD-like DNA helicase ATP-binding domain profile. / DNA helicase, UvrD/REP type / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase beta subunit external 1 domain / RNA polymerase, alpha subunit, C-terminal / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb1, clamp domain superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 2 superfamily / 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, protrusion / RNA polymerase beta subunit / 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 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit alpha / Probable DNA-directed RNA polymerase subunit delta / ATP-dependent DNA helicase rep / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit epsilon / DNA-directed RNA polymerase subunit epsilon / DNA helicase IV / DNA-directed RNA polymerase subunit delta / DNA-directed RNA polymerase subunit alpha ...DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit alpha / Probable DNA-directed RNA polymerase subunit delta / ATP-dependent DNA helicase rep / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit epsilon / DNA-directed RNA polymerase subunit epsilon / DNA helicase IV / DNA-directed RNA polymerase subunit delta / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit beta'
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsPei, H.-P. / Hilal, T. / Huang, Y.-H. / Said, N. / Loll, B. / Wahl, M.C.
Funding support Germany, United States, 6items
OrganizationGrant numberCountry
German Research Foundation (DFG)INST 335/590-1 FUGG Germany
German Research Foundation (DFG)973/C08 Germany
German Research Foundation (DFG)433623608 Germany
German Research Foundation (DFG)2473 Germany
Wellcome Trust103139 United States
Wellcome Trust203149 United States
CitationJournal: Nat Commun / Year: 2020
Title: The δ subunit and NTPase HelD institute a two-pronged mechanism for RNA polymerase recycling.
Authors: Hao-Hong Pei / Tarek Hilal / Zhuo A Chen / Yong-Heng Huang / Yuan Gao / Nelly Said / Bernhard Loll / Juri Rappsilber / Georgiy A Belogurov / Irina Artsimovitch / Markus C Wahl /
Abstract: Cellular RNA polymerases (RNAPs) can become trapped on DNA or RNA, threatening genome stability and limiting free enzyme pools, but how RNAP recycling into active states is achieved remains elusive. ...Cellular RNA polymerases (RNAPs) can become trapped on DNA or RNA, threatening genome stability and limiting free enzyme pools, but how RNAP recycling into active states is achieved remains elusive. In Bacillus subtilis, the RNAP δ subunit and NTPase HelD have been implicated in RNAP recycling. We structurally analyzed Bacillus subtilis RNAP-δ-HelD complexes. HelD has two long arms: a Gre cleavage factor-like coiled-coil inserts deep into the RNAP secondary channel, dismantling the active site and displacing RNA, while a unique helical protrusion inserts into the main channel, prying the β and β' subunits apart and, aided by δ, dislodging DNA. RNAP is recycled when, after releasing trapped nucleic acids, HelD dissociates from the enzyme in an ATP-dependent manner. HelD abundance during slow growth and a dimeric (RNAP-δ-HelD) structure that resembles hibernating eukaryotic RNAP I suggest that HelD might also modulate active enzyme pools in response to cellular cues.
History
DepositionJun 10, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 14, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 13, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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 / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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Assembly

Deposited unit
D: Probable DNA-directed RNA polymerase subunit delta,Probable DNA-directed RNA polymerase subunit delta,Probable DNA-directed RNA polymerase subunit delta
E: RNA polymerase subunit omega
U: DNA-directed RNA polymerase subunit alpha
V: DNA-directed RNA polymerase subunit alpha
X: DNA-directed RNA polymerase subunit beta
Y: DNA-directed RNA polymerase subunit beta'
H: DNA helicase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)451,2388
Polymers451,1737
Non-polymers651
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: light scattering
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area36540 Å2
ΔGint-135 kcal/mol
Surface area163690 Å2
MethodPISA

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Components

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Protein , 3 types, 3 molecules DEH

#1: Protein Probable DNA-directed RNA polymerase subunit delta,Probable DNA-directed RNA polymerase subunit delta,Probable DNA-directed RNA polymerase subunit delta / RNAP delta factor


Mass: 14914.153 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: due to weak electron density, we tentatively traced the C-terminal portion of the protein, but could not assign the sequence,due to weak electron density, we tentatively traced the C- ...Details: due to weak electron density, we tentatively traced the C-terminal portion of the protein, but could not assign the sequence,due to weak electron density, we tentatively traced the C-terminal portion of the protein, but could not assign the sequence,due to weak electron density, we tentatively traced the C-terminal portion of the protein, but could not assign the sequence,due to weak electron density, we tentatively traced the C-terminal portion of the protein, but could not assign the sequence
Source: (natural) Bacillus subtilis (bacteria) / References: UniProt: A0A0D1KIU7, UniProt: P12464*PLUS
#2: Protein RNA polymerase subunit omega /


Mass: 8228.357 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bacillus subtilis (bacteria) / References: UniProt: A0A410WI33, UniProt: O31718*PLUS
#6: Protein DNA helicase / Helicase


Mass: 90052.516 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bacillus subtilis (bacteria)
References: UniProt: A0A164TSE8, UniProt: O32215*PLUS, DNA helicase

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DNA-directed RNA polymerase subunit ... , 3 types, 4 molecules UVXY

#3: Protein DNA-directed RNA polymerase subunit alpha / Polymerase / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 34842.387 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Bacillus subtilis (bacteria)
References: UniProt: A0A063XB83, UniProt: P20429*PLUS, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit beta / Polymerase / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 133847.938 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: DUE TO LIMITED QUALITY OF THE ELECTRON DENSITY, WE WERE ONLY ABLE TO TRACE SOME OF THE PROTEIN MAIN CHAIN AS POLY-ALA
Source: (natural) Bacillus subtilis (bacteria)
References: UniProt: A0A2J0WBQ0, UniProt: P37870*PLUS, DNA-directed RNA polymerase
#5: Protein DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 134444.953 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bacillus subtilis (bacteria)
References: UniProt: A0A063XB23, UniProt: P37871*PLUS, DNA-directed RNA polymerase

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Non-polymers , 1 types, 1 molecules

#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 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: RNA polymerase in complex with HelD / Type: COMPLEX / Entity ID: #1-#6 / Source: NATURAL
Molecular weightValue: 0.46 MDa
Source (natural)Organism: Bacillus subtilis (bacteria)
Buffer solutionpH: 7.6
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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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 magnification: 96000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Image recordingAverage exposure time: 36 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.17.1_3660refinement
PHENIX1.17.1_3660refinement
EM software
IDNameVersionCategory
2EPU2.15image acquisition
10cryoSPARC2.12initial Euler assignment
11cryoSPARC2.14final Euler assignment
12cryoSPARC2.14classification
13cryoSPARC2.143D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 81279 / Symmetry type: POINT
RefinementHighest resolution: 4.2 Å / Cross valid method: NONE
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002629215
ELECTRON MICROSCOPYf_angle_d0.534739465
ELECTRON MICROSCOPYf_chiral_restr0.04194442
ELECTRON MICROSCOPYf_plane_restr0.00365150
ELECTRON MICROSCOPYf_dihedral_angle_d26.50443968

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