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- PDB-8pbl: E. coli RNA polymerase elongation complex stalled at thymine dime... -

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Basic information

Entry
Database: PDB / ID: 8pbl
TitleE. coli RNA polymerase elongation complex stalled at thymine dimer lesion
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • Non-template DNA
  • Template DNA
  • mRNAMessenger RNA
KeywordsTRANSCRIPTION / RNA Polymerase / RNAP / Gene Expression / DNA lesion / Stalling
Function / homology
Function and homology information


RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly / bacterial-type flagellum-dependent cell motility / nitrate assimilation / transcription elongation factor complex / DNA-directed RNA polymerase complex ...RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly / bacterial-type flagellum-dependent cell motility / nitrate assimilation / transcription elongation factor complex / DNA-directed RNA polymerase complex / regulation of DNA-templated transcription elongation / transcription antitermination / cell motility / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat / protein-containing complex assembly / intracellular iron ion homeostasis / protein dimerization activity / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytosol / 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 superfamily / 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 superfamily / 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 / RNA (> 10) / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.87 Å
AuthorsWoodgate, J. / Zenkin, N.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome Trust217189/Z/19/Z United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/M011186/1 United Kingdom
CitationJournal: Nature / Year: 2024
Title: Translation selectively destroys non-functional transcription complexes.
Authors: Jason Woodgate / Hamed Mosaei / Pavel Brazda / Flint Stevenson-Jones / Nikolay Zenkin /
Abstract: Transcription elongation stalls at lesions in the DNA template. For the DNA lesion to be repaired, the stalled transcription elongation complex (EC) has to be removed from the damaged site. Here we ...Transcription elongation stalls at lesions in the DNA template. For the DNA lesion to be repaired, the stalled transcription elongation complex (EC) has to be removed from the damaged site. Here we show that translation, which is coupled to transcription in bacteria, actively dislodges stalled ECs from the damaged DNA template. By contrast, paused, but otherwise elongation-competent, ECs are not dislodged by the ribosome. Instead, they are helped back into processive elongation. We also show that the ribosome slows down when approaching paused, but not stalled, ECs. Our results indicate that coupled ribosomes functionally and kinetically discriminate between paused ECs and stalled ECs, ensuring the selective destruction of only the latter. This functional discrimination is controlled by the RNA polymerase's catalytic domain, the Trigger Loop. We show that the transcription-coupled DNA repair helicase UvrD, proposed to cause backtracking of stalled ECs, does not interfere with ribosome-mediated dislodging. By contrast, the transcription-coupled DNA repair translocase Mfd acts synergistically with translation, and dislodges stalled ECs that were not destroyed by the ribosome. We also show that a coupled ribosome efficiently destroys misincorporated ECs that can cause conflicts with replication. We propose that coupling to translation is an ancient and one of the main mechanisms of clearing non-functional ECs from the genome.
History
DepositionJun 9, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 25, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 24, 2024Group: Database references / Category: citation
Item: _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Feb 14, 2024Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN
Revision 1.3Feb 21, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.4Mar 6, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Non-template DNA
B: Template DNA
D: DNA-directed RNA polymerase subunit alpha
E: DNA-directed RNA polymerase subunit alpha
F: DNA-directed RNA polymerase subunit beta
G: DNA-directed RNA polymerase subunit beta'
H: DNA-directed RNA polymerase subunit omega
R: mRNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)406,20911
Polymers406,0548
Non-polymers1553
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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DNA chain , 2 types, 2 molecules AB

#1: DNA chain Non-template DNA


Mass: 15107.696 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#2: DNA chain Template DNA


Mass: 15384.848 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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

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


Mass: 26487.205 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoA, FTV90_00720, FTV93_20930 / Production host: Escherichia coli (E. coli)
References: UniProt: A0A5B9AW69, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit beta / Polymerase / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 150820.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoB, Z5560, ECs4910 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A8V4, DNA-directed RNA polymerase
#5: Protein DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 155366.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoC, Z5561, ECs4911 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A8T8, DNA-directed RNA polymerase
#6: Protein DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 10249.547 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoZ, b3649, JW3624 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A800, DNA-directed RNA polymerase

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RNA chain , 1 types, 1 molecules R

#7: RNA chain mRNA / Messenger RNA


Mass: 6149.746 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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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 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: E. coli RNA Polymerase stalled at T=T lesion. / Type: COMPLEX / Entity ID: #1-#7 / Source: RECOMBINANT
Molecular weightValue: 0.5 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Positive Charge / Grid 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: 277.15 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 240000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 3.2 sec. / Electron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 16264

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Processing

EM software
IDNameVersionCategory
4RELION4CTF correction
7UCSF ChimeraX1.5model fitting
9PHENIX1.2model refinement
10Coot0.9.8.4model refinement
14RELION43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.87 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 131098 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDAccession codeDetailsInitial refinement model-ID
18FVR

8fvr

8FVRRNAP Subunits1
21SL2

1sl2

1SL2T=T Lesion2

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