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Yorodumi- PDB-8g8z: Cryo-EM structure of 3DVA component 1 of Escherichia coli que-PEC... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8g8z | |||||||||||||||
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Title | Cryo-EM structure of 3DVA component 1 of Escherichia coli que-PEC (paused elongation complex) RNA Polymerase plus preQ1 ligand | |||||||||||||||
Components |
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Keywords | TRANSCRIPTION / Polymerase / RNAP / riboswitch / aptamer / PreQ1 | |||||||||||||||
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 / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm Similarity search - Function | |||||||||||||||
Biological species | Escherichia coli (E. coli) | |||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 Å | |||||||||||||||
Authors | Porta, J.C. / Ohi, M.D. / Walter, N.G. / Frank, A.T. / Deb, I. / Meze, K. | |||||||||||||||
Funding support | United States, 4items
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Citation | Journal: 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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8g8z.cif.gz | 606.8 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8g8z.ent.gz | 483.9 KB | Display | PDB format |
PDBx/mmJSON format | 8g8z.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/g8/8g8z ftp://data.pdbj.org/pub/pdb/validation_reports/g8/8g8z | HTTPS FTP |
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-Related structure data
Related structure data | 29859MC 8f3cC 8g00C 8g1sC 8g2wC 8g4wC 8g7eC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-DNA chain , 2 types, 2 molecules AB
#1: DNA chain | 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) |
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#2: DNA chain | 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) |
-DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules GHKIJ
#3: Protein | Mass: 25971.531 Da / Num. of mol.: 2 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, DNA-directed RNA polymerase #4: Protein | | Mass: 9094.239 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: A0A1X3IVJ5, DNA-directed RNA polymerase #6: Protein | | Mass: 150560.562 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: C3SIA7 #7: Protein | | Mass: 150436.344 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: A0A369F490 |
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-RNA chain , 1 types, 1 molecules R
#5: RNA chain | Mass: 15404.284 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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-Non-polymers , 2 types, 2 molecules
#8: Chemical | ChemComp-PRF / |
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#9: Chemical | ChemComp-MG / |
-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Cryo-EM structure of 3DVA component 1 of Escherichia coli que-PEC (paused elongation complex) RNA Polymerase plus preQ1 ligand Type: COMPLEX / Entity ID: #1-#7 / Source: RECOMBINANT |
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Molecular weight | Value: 0.42 MDa / Experimental value: NO |
Source (natural) | Organism: Escherichia coli (E. coli) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % Details: Vitrification was carried out in a chamber with the temperature set to 4 C. |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3500 nm / Nominal defocus min: 500 nm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 62 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
Image scans | Width: 1 / Height: 1 / Movie frames/image: 40 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Particle selection | Details: This is component 1 of 3DVA analysis of a particle stack of Cryo-EM consensus structure of Escherichia coli que-PEC (paused elongation complex) RNA Polymerase plus preQ1 ligand. Particles were already selected. | ||||||||||||||||||||||||
3D reconstruction | Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 20469 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross-correlation coefficient | ||||||||||||||||||||||||
Atomic model building | PDB-ID: 6ASX Accession code: 6ASX / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||
Refinement | Highest resolution: 4.3 Å | ||||||||||||||||||||||||
Refine LS restraints |
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