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- PDB-6wvj: Cryo-EM structure of Bacillus subtilis RNA Polymerase elongation ... -

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Basic information

Entry
Database: PDB / ID: 6wvj
TitleCryo-EM structure of Bacillus subtilis RNA Polymerase elongation complex
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • DNA (5'-D(*TP*GP*TP*CP*GP*GP*GP*CP*GP*TP*CP*CP*GP*CP*GP*CP*GP*CP*C)-3')
  • DNA (5'-D(P*AP*CP*GP*CP*CP*CP*GP*AP*CP*A)-3')
  • RNA (5'-R(P*GP*GP*CP*GP*CP*GP*CP*G)-3')
KeywordsTRANSCRIPTION/DNA/RNA / DNA-DEPENDENT RNA POLYMERASE / TRANSCRIPTION / TRANSCRIPTION-DNA-RNA COMPLEX
Function / homology
Function and homology information


DNA-directed RNA polymerase complex / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / response to antibiotic / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm
Similarity search - Function
: / DNA-directed RNA polymerase, omega subunit / 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 Rpb6 ...: / DNA-directed RNA polymerase, omega subunit / 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 Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb1, clamp domain superfamily / RPB6/omega subunit-like 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
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit omega / 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)
Escherichia coli BL21 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.36 Å
AuthorsNewing, T. / Tolun, G. / Oakley, A.J.
CitationJournal: Nat Commun / Year: 2020
Title: Molecular basis for RNA polymerase-dependent transcription complex recycling by the helicase-like motor protein HelD.
Authors: Timothy P Newing / Aaron J Oakley / Michael Miller / Catherine J Dawson / Simon H J Brown / James C Bouwer / Gökhan Tolun / Peter J Lewis /
Abstract: In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions. Gram- ...In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions. Gram-positive bacteria contain a transcription factor HelD that is able to remove and recycle stalled complexes, but it was not known how it performed this function. Here, using single particle cryo-electron microscopy, we have determined the structures of Bacillus subtilis RNA polymerase (RNAP) elongation and HelD complexes, enabling analysis of the conformational changes that occur in RNAP driven by HelD interaction. HelD has a 2-armed structure which penetrates deep into the primary and secondary channels of RNA polymerase. One arm removes nucleic acids from the active site, and the other induces a large conformational change in the primary channel leading to removal and recycling of the stalled polymerase, representing a novel mechanism for recycling transcription complexes in bacteria.
History
DepositionMay 6, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 18, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 6, 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.name
Revision 1.2Nov 23, 2022Group: Database references / Category: database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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  • Deposited structure unit
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  • EMDB-21920
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Assembly

Deposited unit
A: DNA-directed RNA polymerase subunit alpha
B: DNA-directed RNA polymerase subunit alpha
C: DNA-directed RNA polymerase subunit beta
D: DNA-directed RNA polymerase subunit beta'
F: DNA-directed RNA polymerase subunit omega
T: DNA (5'-D(*TP*GP*TP*CP*GP*GP*GP*CP*GP*TP*CP*CP*GP*CP*GP*CP*GP*CP*C)-3')
R: RNA (5'-R(P*GP*GP*CP*GP*CP*GP*CP*G)-3')
N: DNA (5'-D(P*AP*CP*GP*CP*CP*CP*GP*AP*CP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)357,31011
Polymers357,1558
Non-polymers1553
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area32970 Å2
ΔGint-193 kcal/mol
Surface area114970 Å2

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Components

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DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules ABCDF

#1: 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 genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (strain 168) (bacteria)
Strain: 168 / Gene: rpoA, BSU01430 / Plasmid: pNG1256 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P20429, DNA-directed RNA polymerase
#2: 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 genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria)
Gene: rpoB, A3772_00695, BS16045_00134, ETA10_00685, ETK61_00685, GII79_00680
Plasmid: pNG1256 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
References: UniProt: A0A2J0WBQ0, UniProt: P37870*PLUS, DNA-directed RNA polymerase
#3: 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 genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria)
Gene: rpoC, A3772_00700, B4122_1444, B4417_1498, BS16045_00135, ETA10_00690, ETK61_00690, GII79_00685
Plasmid: pNG1256 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
References: UniProt: A0A063XB23, UniProt: P37871*PLUS, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 7766.921 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria)
Gene: rpoZ, A3772_08560, AMC98_03500, B4122_2750, B4417_3585, BS16045_01675, DFO69_2436, ETA10_08595, ETK61_08870, FIU26_02565, FZC71_02355, GII79_08370, SC09_Contig19orf01098
Plasmid: pNG1256 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
References: UniProt: A0A063XI46, UniProt: O35011*PLUS, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules TN

#5: DNA chain DNA (5'-D(*TP*GP*TP*CP*GP*GP*GP*CP*GP*TP*CP*CP*GP*CP*GP*CP*GP*CP*C)-3')


Mass: 5814.721 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
#7: DNA chain DNA (5'-D(P*AP*CP*GP*CP*CP*CP*GP*AP*CP*A)-3')


Mass: 2998.984 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)

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RNA chain , 1 types, 1 molecules R

#6: RNA chain RNA (5'-R(P*GP*GP*CP*GP*CP*GP*CP*G)-3')


Mass: 2596.617 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)

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Non-polymers , 2 types, 3 molecules

#8: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#9: Chemical ChemComp-MG / MAGNESIUM ION


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

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Details

Has ligand of interestN
Sequence detailsThe authors state that the DNA and RNA sequences were built de novo based on density.

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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
1DNA-directed RNA polymerase elongation complexCOMPLEX#1-#70MULTIPLE SOURCES
2DNA-directed RNA polymerase subunit alpha, DNA-directed RNA polymerase subunit beta, DNA-directed RNA polymerase subunit beta', DNA-directed RNA polymerase subunit omega/DNA ComplexPolymeraseCOMPLEX#1-#41RECOMBINANT
3DNA, RNACOMPLEX#5-#71NATURAL
Molecular weightValue: 0.344200 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Bacillus subtilis (bacteria)1423
23Escherichia coli BL21(DE3) (bacteria)469008
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pNG1256
Buffer solutionpH: 7.8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisNH2C(CH2OH)31
2150 mMSodium ChlorideNaClSodium chloride1
310 mMMagnesium ChlorideMgCl21
41 mMEDTAEthylenediaminetetraacetic acid(HO2CCH2)2NCH2CH2N(CH2CO2H)21
55 mMDTTHSCH2CH(OH)CH(OH)CH2SH1
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K
Details: Sample loading volume ranged between 2 and 3 microlitres. Samples were blotted for 5 seconds prior to vitrification.

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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 / Calibrated magnification: 59500 X / Nominal defocus max: 2800 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9 sec. / Electron dose: 52.2 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 5185
EM imaging opticsEnergyfilter name: GIF Quantum LS
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.18rc3_3805: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4GctfCTF correction
9PHENIXmodel refinement
10ISOLDEmodel refinement
11RELION3.1initial Euler assignment
12RELION3.1final Euler assignment
13RELION3.1classification
14RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 463519
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.36 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 58854 / Symmetry type: POINT
Atomic model buildingSpace: REAL
RefinementHighest resolution: 3.36 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00422812
ELECTRON MICROSCOPYf_angle_d0.58631054
ELECTRON MICROSCOPYf_dihedral_angle_d18.8313425
ELECTRON MICROSCOPYf_chiral_restr0.0433583
ELECTRON MICROSCOPYf_plane_restr0.0043918

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