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Yorodumi- PDB-8fix: Cryo-EM structure of E. coli RNA polymerase backtracked elongatio... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8fix | |||||||||
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Title | Cryo-EM structure of E. coli RNA polymerase backtracked elongation complex harboring a terminal mismatch | |||||||||
Components |
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Keywords | TRANSCRIPTION / RNA polymerase / ribosome / coupling | |||||||||
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 | |||||||||
Biological species | Escherichia coli K-12 (bacteria) Escherichia phage Lambda (virus) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å | |||||||||
Authors | Florez Ariza, A. / Wee, L. / Tong, A. / Canari, C. / Grob, P. / Nogales, E. / Bustamante, C. | |||||||||
Funding support | United States, 2items
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Citation | Journal: Cell / Year: 2023 Title: A trailing ribosome speeds up RNA polymerase at the expense of transcript fidelity via force and allostery. Authors: Liang Meng Wee / Alexander B Tong / Alfredo Jose Florez Ariza / Cristhian Cañari-Chumpitaz / Patricia Grob / Eva Nogales / Carlos J Bustamante / Abstract: In prokaryotes, translation can occur on mRNA that is being transcribed in a process called coupling. How the ribosome affects the RNA polymerase (RNAP) during coupling is not well understood. Here, ...In prokaryotes, translation can occur on mRNA that is being transcribed in a process called coupling. How the ribosome affects the RNA polymerase (RNAP) during coupling is not well understood. Here, we reconstituted the E. coli coupling system and demonstrated that the ribosome can prevent pausing and termination of RNAP and double the overall transcription rate at the expense of fidelity. Moreover, we monitored single RNAPs coupled to ribosomes and show that coupling increases the pause-free velocity of the polymerase and that a mechanical assisting force is sufficient to explain the majority of the effects of coupling. Also, by cryo-EM, we observed that RNAPs with a terminal mismatch adopt a backtracked conformation, while a coupled ribosome allosterically induces these polymerases toward a catalytically active anti-swiveled state. Finally, we demonstrate that prolonged RNAP pausing is detrimental to cell viability, which could be prevented by polymerase reactivation through a coupled ribosome. | |||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8fix.cif.gz | 591.3 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8fix.ent.gz | 469.3 KB | Display | PDB format |
PDBx/mmJSON format | 8fix.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8fix_validation.pdf.gz | 1.2 MB | Display | wwPDB validaton report |
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Full document | 8fix_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 8fix_validation.xml.gz | 94.3 KB | Display | |
Data in CIF | 8fix_validation.cif.gz | 146.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/fi/8fix ftp://data.pdbj.org/pub/pdb/validation_reports/fi/8fix | HTTPS FTP |
-Related structure data
Related structure data | 29212MC 8fiyC 8fizC 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 NT
#1: DNA chain | Mass: 4531.978 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia phage Lambda (virus) |
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#2: DNA chain | Mass: 7158.604 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia phage Lambda (virus) |
-DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules ABCDE
#3: Protein | Mass: 36558.680 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: rpoA, pez, phs, sez, b3295, JW3257 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P0A7Z4, DNA-directed RNA polymerase #4: Protein | | Mass: 150820.875 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 (bacteria) Gene: rpoB, groN, nitB, rif, ron, stl, stv, tabD, b3987, JW3950 Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P0A8V2, DNA-directed RNA polymerase #5: Protein | | Mass: 155366.781 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: rpoC, tabB, b3988, JW3951 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P0A8T7, DNA-directed RNA polymerase #7: Protein | | Mass: 10249.547 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: rpoZ, b3649, JW3624 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P0A800, DNA-directed RNA polymerase |
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-RNA chain , 1 types, 1 molecules R
#6: RNA chain | Mass: 3467.114 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia phage Lambda (virus) |
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-Non-polymers , 2 types, 3 molecules
#8: Chemical | #9: Chemical | ChemComp-MG / | |
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-Details
Has ligand of interest | N |
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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 |
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Molecular weight | Value: 0.4 MDa / Experimental value: NO | ||||||||||||||||||||||||||||||
Source (natural) |
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Source (recombinant) | Organism: Escherichia coli BL21 (bacteria) | ||||||||||||||||||||||||||||||
Buffer solution | pH: 7.5 | ||||||||||||||||||||||||||||||
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||
Specimen support | Grid type: C-flat-1.2/1.3 | ||||||||||||||||||||||||||||||
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.18.2_3874: / Classification: refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 118450 / Symmetry type: POINT | ||||||||||||||||||||||||
Refine LS restraints |
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