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- PDB-7yp9: Cryo-EM structure of Escherichia coli paused complex of transcrip... -

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Basic information

Entry
Database: PDB / ID: 7yp9
TitleCryo-EM structure of Escherichia coli paused complex of transcription termination (TTC-pause)
Components
  • (DNA (31-MER)) x 2
  • (DNA-directed RNA polymerase subunit ...) x 4
  • RNA (5'-R(*GP*CP*GP*UP*CP*GP*CP*AP*GP*GP*CP*CP*UP*UP*UP*UP*UP*AP*UP*U)-3')
KeywordsTRANSCRIPTION/DNA/RNA / Termination / Intrinsic termination / U-tract pause / Transcription pause / Factor-independent termination / TRANSCRIPTION-DNA-RNA COMPLEX
Function / homology
Function and homology information


RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / bacterial-type RNA polymerase core enzyme binding / 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 ...RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / bacterial-type RNA polymerase core enzyme binding / 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 / regulation of DNA-templated transcription elongation / DNA-templated transcription initiation / transcription antitermination / cell motility / 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 / response to antibiotic / magnesium ion binding / DNA binding / zinc ion binding / membrane / 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 / RPB6/omega subunit-like superfamily / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / 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 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / 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 K-12 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.58 Å
AuthorsYou, L.L. / Zhang, Y.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Nature / Year: 2023
Title: Structural basis for intrinsic transcription termination.
Authors: Linlin You / Expery O Omollo / Chengzhi Yu / Rachel A Mooney / Jing Shi / Liqiang Shen / Xiaoxian Wu / Aijia Wen / Dingwei He / Yuan Zeng / Yu Feng / Robert Landick / Yu Zhang /
Abstract: Efficient and accurate termination is required for gene transcription in all living organisms. Cellular RNA polymerases in both bacteria and eukaryotes can terminate their transcription through a ...Efficient and accurate termination is required for gene transcription in all living organisms. Cellular RNA polymerases in both bacteria and eukaryotes can terminate their transcription through a factor-independent termination pathway-called intrinsic termination transcription in bacteria-in which RNA polymerase recognizes terminator sequences, stops nucleotide addition and releases nascent RNA spontaneously. Here we report a set of single-particle cryo-electron microscopy structures of Escherichia coli transcription intrinsic termination complexes representing key intermediate states of the event. The structures show how RNA polymerase pauses at terminator sequences, how the terminator RNA hairpin folds inside RNA polymerase, and how RNA polymerase rewinds the transcription bubble to release RNA and then DNA. These macromolecular snapshots define a structural mechanism for bacterial intrinsic termination and a pathway for RNA release and DNA collapse that is relevant for factor-independent termination by all RNA polymerases.
History
DepositionAug 3, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 16, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 18, 2023Group: Database references / Refinement description
Category: citation / citation_author / pdbx_initial_refinement_model
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Jan 25, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.3Feb 8, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.4Jul 3, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin
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 / _em_admin.last_update

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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 alpha
B: DNA-directed RNA polymerase subunit alpha
C: DNA-directed RNA polymerase subunit beta
D: DNA-directed RNA polymerase subunit beta'
E: DNA-directed RNA polymerase subunit omega
F: DNA (31-MER)
G: DNA (31-MER)
H: RNA (5'-R(*GP*CP*GP*UP*CP*GP*CP*AP*GP*GP*CP*CP*UP*UP*UP*UP*UP*AP*UP*U)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)416,39411
Polymers416,2398
Non-polymers1553
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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

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


Mass: 36558.680 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K-12 / Gene: rpoA / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0A7Z4, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / 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 K-12 (bacteria) / Strain: K-12 / Gene: rpoB / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0A8V2, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 156537.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K-12 / Gene: rpoC / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0A8T7, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / 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 K-12 (bacteria) / Strain: K-12 / Gene: rpoZ / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P0A800, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules FG

#5: DNA chain DNA (31-MER)


Mass: 9573.204 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: DNA chain DNA (31-MER)


Mass: 9626.211 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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RNA chain , 1 types, 1 molecules H

#7: RNA chain RNA (5'-R(*GP*CP*GP*UP*CP*GP*CP*AP*GP*GP*CP*CP*UP*UP*UP*UP*UP*AP*UP*U)-3')


Mass: 6314.720 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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

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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
1E. coli paused complex of transcription terminationCOMPLEX#1-#70MULTIPLE SOURCES
2DNA-directed RNA polymeraseCOMPLEX#1-#41RECOMBINANT
3DNA/RNACOMPLEX#5-#71SYNTHETIC
Molecular weightValue: 0.4 MDa / Experimental value: YES
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K-12
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
Buffer solutionpH: 7.5
SpecimenConc.: 13.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / 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 FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 8 sec. / Electron dose: 60.8 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1796
Image scansMovie frames/image: 38

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Processing

EM software
IDNameVersionCategory
1RELION3.08particle selection
2SerialEMimage acquisition
4CTFFIND4CTF correction
7UCSF Chimera1.10.2model fitting
9PHENIX1.14model refinement
10RELION3.08initial Euler assignment
11RELION3.08final Euler assignment
12RELION3.08classification
13RELION3.083D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 203216
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.58 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 132648 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6ALF

6alf


Accession code: 6ALF / Source name: PDB / Type: experimental model

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