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Yorodumi- PDB-8ac1: RNA polymerase at U-rich pause bound to non-regulatory RNA - inac... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8ac1 | |||||||||||||||
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Title | RNA polymerase at U-rich pause bound to non-regulatory RNA - inactive, open clamp state | |||||||||||||||
Components |
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Keywords | TRANSCRIPTION / RNA polymerase / transcriptional pausing / transcription termination / regulatory RNA | |||||||||||||||
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 / transcription antitermination / DNA-templated transcription initiation / 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 | |||||||||||||||
Biological species | Escherichia coli K-12 (bacteria) Escherichia coli (E. coli) | |||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.06 Å | |||||||||||||||
Authors | Dey, S. / Weixlbaumer, A. | |||||||||||||||
Funding support | European Union, France, 4items
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Citation | Journal: Mol Cell / Year: 2022 Title: Structural insights into RNA-mediated transcription regulation in bacteria. Authors: Sanjay Dey / Claire Batisse / Jinal Shukla / Michael W Webster / Maria Takacs / Charlotte Saint-André / Albert Weixlbaumer / Abstract: RNA can regulate its own synthesis without auxiliary proteins. For example, U-rich RNA sequences signal RNA polymerase (RNAP) to pause transcription and are required for transcript release at ...RNA can regulate its own synthesis without auxiliary proteins. For example, U-rich RNA sequences signal RNA polymerase (RNAP) to pause transcription and are required for transcript release at intrinsic terminators in all kingdoms of life. In contrast, the regulatory RNA putL suppresses pausing and termination in cis. However, how nascent RNA modulates its own synthesis remains largely unknown. We present cryo-EM reconstructions of RNAP captured during transcription of putL variants or an unrelated sequence at a U-rich pause site. Our results suggest how putL suppresses pausing and promotes its synthesis. We demonstrate that transcribing a U-rich sequence, a ubiquitous trigger of intrinsic termination, promotes widening of the RNAP nucleic-acid-binding channel. Widening destabilizes RNAP interactions with DNA and RNA to facilitate transcript dissociation reminiscent of intrinsic transcription termination. Surprisingly, RNAP remains bound to DNA after transcript release. Our results provide the structural framework to understand RNA-mediated intrinsic transcription termination. | |||||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8ac1.cif.gz | 602.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8ac1.ent.gz | 459.2 KB | Display | PDB format |
PDBx/mmJSON format | 8ac1.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8ac1_validation.pdf.gz | 1.6 MB | Display | wwPDB validaton report |
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Full document | 8ac1_full_validation.pdf.gz | 1.7 MB | Display | |
Data in XML | 8ac1_validation.xml.gz | 100.3 KB | Display | |
Data in CIF | 8ac1_validation.cif.gz | 148.6 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ac/8ac1 ftp://data.pdbj.org/pub/pdb/validation_reports/ac/8ac1 | HTTPS FTP |
-Related structure data
Related structure data | 15330MC 8abyC 8abzC 8ac0C 8ac2C 8acpC 8ad1C 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-directed RNA polymerase subunit ... , 4 types, 5 molecules ABCDE
#1: 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(DE3) (bacteria) / References: UniProt: P0A7Z4, DNA-directed RNA polymerase #2: 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(DE3) (bacteria) / References: UniProt: P0A8V2, DNA-directed RNA polymerase #3: Protein | | Mass: 155237.672 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: rpoC, Z5561, ECs4911 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A8T8, DNA-directed RNA polymerase #4: 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(DE3) (bacteria) / References: UniProt: P0A800, DNA-directed RNA polymerase |
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-DNA chain , 2 types, 2 molecules NT
#5: DNA chain | Mass: 91015.141 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli) |
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#7: DNA chain | Mass: 91180.203 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli) |
-RNA chain , 1 types, 1 molecules R
#6: RNA chain | Mass: 39226.262 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-MG / |
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#9: Chemical |
-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: Escherichia coli RNA polymerase non-regulatory RNA complex - Inactive, open clamp state Type: COMPLEX / Entity ID: #1-#7 / Source: RECOMBINANT |
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Molecular weight | Value: 0.435 MDa / Experimental value: NO |
Source (natural) | Organism: Escherichia coli K-12 (bacteria) |
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) |
Buffer solution | pH: 8 Details: 20 mM Tris-glutamate pH 8.0, 50 mM K-glutamate, 10 mM Mg-glutamate, 0.001 mM ZnCl2, 2mM DTT |
Specimen | Conc.: 12 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: The co-transcriptionally halted complexes for cryo-EM analysis were prepared in vitro by mixing E. coli RNAP holoenzyme with templates containing the DNA base analogue isoG. The RNAP ECs ...Details: The co-transcriptionally halted complexes for cryo-EM analysis were prepared in vitro by mixing E. coli RNAP holoenzyme with templates containing the DNA base analogue isoG. The RNAP ECs halted at the U-rich pause (G122). The complexes were prepared in 0.03 ml reactions and contained: 0.02 mM of E. coli RNAP, 0.080 mM of E. coli Sigma70, 0.02 mM of template DNA, 20 mM Tris-glutamate pH 8.0, 50 mM K-glutamate, 10 mM Mg-glutamate, 0.001 mM ZnCl2, 2mM DTT and 6 mM of each ATP, GTP, CTP and UTP. Initially, all the components except NTPs were added and incubated for 10 min at 310K. To allow transcription elongation, NTPs were added and the sample was incubated for 20 to 40 min at 310K. The final products were purified on a Superose 6 Increase 3.2/300 gel filtration column equilibrated in Glutamate buffer. Samples were concentrated to 10-12 mg/mL using an Amicon Ultra 0.5mL centrifugal filter unit (30 KDa MWCO). Before grid freezing, 8 mM of CHAPSO was added to freshly prepared sample to overcome preferred particle orientation. |
Specimen support | Grid material: GOLD / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283 K Details: Quantifoil UltrAuFoil R1.2/1.3 300 mesh holey gold grids were plasma cleaned on a Model 1070 (Fischione Instruments) for 30 sec at 70% power and with an 80% Argon and 20% Oxygen mixture ...Details: Quantifoil UltrAuFoil R1.2/1.3 300 mesh holey gold grids were plasma cleaned on a Model 1070 (Fischione Instruments) for 30 sec at 70% power and with an 80% Argon and 20% Oxygen mixture prior to the application of 0.004 ml of sample. Grids were plunge frozen into liquid ethane using a Vitrobot mark IV (FEI) with 95% chamber humidity at 283K. |
-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 FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm |
Specimen holder | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 2.2 sec. / Electron dose: 52 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 |
-Processing
Software |
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 4.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 25100 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: map cross correlation | ||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6ALH Accession code: 6ALH / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 147.24 Å2 | ||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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