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- PDB-8g00: Cryo-EM structure of 3DVA component 0 of Escherichia coli que-PEC... -

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Basic information

Entry
Database: PDB / ID: 8g00
TitleCryo-EM structure of 3DVA component 0 of Escherichia coli que-PEC (paused elongation complex) RNA Polymerase minus preQ1 ligand
Components
  • (DNA-directed RNA polymerase subunit ...) x 5
  • DNA (31-MER)
  • DNA (39-mer)
  • RNA (47-MER)
KeywordsTRANSCRIPTION / RNA Polymerase / Riboswitch / preQ1
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 / DNA-directed RNA polymerase 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 / DNA-directed RNA polymerase complex / 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 / DNA-templated transcription / 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 beta' / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsPorta, J.C. / Chauvier, A. / Deb, I. / Ellinger, E. / Frank, A.T. / Meze, K. / Ohi, M.D. / Walter, N.G.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131922 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118524 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD020011 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD030275 United States
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Structural basis for control of bacterial RNA polymerase pausing by a riboswitch and its ligand.
Authors: Adrien Chauvier / Jason C Porta / Indrajit Deb / Emily Ellinger / Katarina Meze / Aaron T Frank / Melanie D Ohi / Nils G Walter /
Abstract: Folding of nascent transcripts can be modulated by the RNA polymerase (RNAP) that carries out their transcription, and vice versa. A pause of RNAP during transcription of a preQ riboswitch (termed ...Folding of nascent transcripts can be modulated by the RNA polymerase (RNAP) that carries out their transcription, and vice versa. A pause of RNAP during transcription of a preQ riboswitch (termed que-PEC) is stabilized by a previously characterized template consensus sequence and the ligand-free conformation of the nascent RNA. Ligand binding to the riboswitch induces RNAP pause release and downstream transcription termination; however, the mechanism by which riboswitch folding modulates pausing is unclear. Here, we report single-particle cryo-electron microscopy reconstructions of que-PEC in ligand-free and ligand-bound states. In the absence of preQ, the RNA transcript is in an unexpected hyper-translocated state, preventing downstream nucleotide incorporation. Strikingly, on ligand binding, the riboswitch rotates around its helical axis, expanding the surrounding RNAP exit channel and repositioning the transcript for elongation. Our study reveals the tight coupling by which nascent RNA structures and their ligands can functionally regulate the macromolecular transcription machinery.
History
DepositionJan 31, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 21, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 26, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jun 19, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / em_3d_fitting_list
Item: _em_3d_fitting_list.initial_refinement_model_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA (39-mer)
B: DNA (31-MER)
G: DNA-directed RNA polymerase subunit alpha
H: DNA-directed RNA polymerase subunit alpha
I: DNA-directed RNA polymerase subunit beta
J: DNA-directed RNA polymerase subunit beta'
K: DNA-directed RNA polymerase subunit omega
R: RNA (47-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)408,7949
Polymers408,7698
Non-polymers241
Water00
1


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

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Components

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DNA chain , 2 types, 2 molecules AB

#1: DNA chain DNA (39-mer)


Mass: 12063.754 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli)
#2: DNA chain DNA (31-MER)


Mass: 9486.079 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli)

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DNA-directed RNA polymerase subunit ... , 5 types, 5 molecules GHIJK

#3: Protein DNA-directed RNA polymerase subunit alpha


Mass: 25999.611 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoA / Production host: Escherichia coli (E. coli) / References: UniProt: A0A5B9AW69
#4: Protein DNA-directed RNA polymerase subunit alpha


Mass: 26487.205 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoA / Production host: Escherichia coli (E. coli) / References: UniProt: A0A5B9AW69
#5: 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 (E. coli) / Gene: rpoB / Production host: Escherichia coli (E. coli) / References: UniProt: P0A8V2, DNA-directed RNA polymerase
#6: Protein DNA-directed RNA polymerase subunit beta'


Mass: 158603.297 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoC / Production host: Escherichia coli (E. coli) / References: UniProt: C3SIA2
#7: 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 (E. coli) / Gene: rpoZ / Production host: Escherichia coli (E. coli) / References: UniProt: P0A800, DNA-directed RNA polymerase

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RNA chain / Non-polymers , 2 types, 2 molecules R

#8: RNA chain RNA (47-MER)


Mass: 15059.079 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli)
#9: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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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: Cryo-EM structure of 3DVA component 0 of Escherichia coli que-PEC (paused elongation complex) RNA Polymerase minus preQ1 ligand .
Type: COMPLEX
Details: Component 0 conformation generated by 3D variability analysis (3DVA) in cryoSPARC
Entity ID: #1-#8 / Source: RECOMBINANT
Molecular weightValue: 0.420 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mg/mLTRIS-HCLC4H11NO31
2100 mMPotassium ChlorideKCl1
31 mMMagnesium chlorideMgCl21
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationCryogen name: ETHANE
Details: VITRIFICATION WAS CARRIED OUT IN A CHAMBER WITH THE TEMPERATURE SET TO 4 DEGREES CELSIUS.

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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: 3500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 62 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansWidth: 1 / Height: 1 / Movie frames/image: 40

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionDetails: This is component 0 of 3DVA analysis of a particle stack of the Cryo-EM consensus structure of Escherichia coli que-PEC (paused elongation complex) RNA Polymerase minus preQ1 ligand. ...Details: This is component 0 of 3DVA analysis of a particle stack of the Cryo-EM consensus structure of Escherichia coli que-PEC (paused elongation complex) RNA Polymerase minus preQ1 ligand. Particles were already selected.
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 57767 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Atomic model buildingPDB-ID: 6ALF

6alf


Accession code: 6ALF / Source name: PDB / Type: experimental model
RefinementHighest resolution: 3.4 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00327465
ELECTRON MICROSCOPYf_angle_d0.57737538
ELECTRON MICROSCOPYf_dihedral_angle_d14.2844533
ELECTRON MICROSCOPYf_chiral_restr0.0434323
ELECTRON MICROSCOPYf_plane_restr0.0054561

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