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Yorodumi- PDB-8u3b: Cryo-EM structure of E. coli NarL-transcription activation comple... -
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-Basic information
Entry | Database: PDB / ID: 8u3b | ||||||
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Title | Cryo-EM structure of E. coli NarL-transcription activation complex at 3.2A | ||||||
Components |
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Keywords | transcription/DNA / transcription / RNA polymerase / transcription activation / NarL / nitrate-responsive regulator / transcription-DNA complex | ||||||
Function / homology | Function and homology information sigma factor antagonist complex / DNA-binding transcription repressor activity / 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 / sigma factor activity / bacterial-type flagellum assembly ...sigma factor antagonist complex / DNA-binding transcription repressor activity / 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 / sigma factor activity / bacterial-type flagellum assembly / phosphorelay signal transduction system / bacterial-type flagellum-dependent cell motility / nitrate assimilation / transcription elongation factor complex / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription initiation / protein-DNA complex / 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 / transcription cis-regulatory region binding / response to antibiotic / negative regulation of DNA-templated transcription / regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / ATP binding / identical protein binding / membrane / cytosol / cytoplasm Similarity search - Function | ||||||
Biological species | Escherichia coli (E. coli) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.23 Å | ||||||
Authors | Liu, B. / Kompaniiets, D. / Wang, D. | ||||||
Funding support | 1items
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Citation | Journal: Nucleic Acids Res / Year: 2024 Title: Structural basis for transcription activation by the nitrate-responsive regulator NarL. Authors: Dmytro Kompaniiets / Lina He / Dong Wang / Wei Zhou / Yang Yang / Yangbo Hu / Bin Liu / Abstract: Transcription activation is a crucial step of regulation during transcription initiation and a classic check point in response to different stimuli and stress factors. The Escherichia coli NarL is a ...Transcription activation is a crucial step of regulation during transcription initiation and a classic check point in response to different stimuli and stress factors. The Escherichia coli NarL is a nitrate-responsive global transcription factor that controls the expression of nearly 100 genes. However, the molecular mechanism of NarL-mediated transcription activation is not well defined. Here we present a cryo-EM structure of NarL-dependent transcription activation complex (TAC) assembled on the yeaR promoter at 3.2 Å resolution. Our structure shows that the NarL dimer binds at the -43.5 site of the promoter DNA with its C-terminal domain (CTD) not only binding to the DNA but also making interactions with RNA polymerase subunit alpha CTD (αCTD). The key role of these NarL-mediated interactions in transcription activation was further confirmed by in vivo and in vitro transcription assays. Additionally, the NarL dimer binds DNA in a different plane from that observed in the structure of class II TACs. Unlike the canonical class II activation mechanism, NarL does not interact with σ4, while RNAP αCTD is bound to DNA on the opposite side of NarL. Our findings provide a structural basis for detailed mechanistic understanding of NarL-dependent transcription activation on yeaR promoter and reveal a potentially novel mechanism of transcription activation. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
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PDBx/mmCIF format | 8u3b.cif.gz | 752.9 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8u3b.ent.gz | 600.5 KB | Display | PDB format |
PDBx/mmJSON format | 8u3b.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8u3b_validation.pdf.gz | 1.4 MB | Display | wwPDB validaton report |
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Full document | 8u3b_full_validation.pdf.gz | 1.5 MB | Display | |
Data in XML | 8u3b_validation.xml.gz | 114.9 KB | Display | |
Data in CIF | 8u3b_validation.cif.gz | 180.4 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/u3/8u3b ftp://data.pdbj.org/pub/pdb/validation_reports/u3/8u3b | HTTPS FTP |
-Related structure data
Related structure data | 41856MC 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 (E. coli) / Gene: rpoA, pez, phs, sez, b3295, JW3257 / Production host: Escherichia coli (E. coli) / 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 (E. coli) Gene: rpoB, groN, nitB, rif, ron, stl, stv, tabD, b3987, JW3950 Production host: Escherichia coli (E. coli) / References: UniProt: P0A8V2, DNA-directed RNA polymerase #3: Protein | | Mass: 155366.781 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoC, tabB, b3988, JW3951 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A8T7, 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 (E. coli) / Gene: rpoZ, b3649, JW3624 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A800, DNA-directed RNA polymerase |
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-Protein , 2 types, 3 molecules FGH
#5: Protein | Mass: 72206.266 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoD, alt, b3067, JW3039 / Production host: Escherichia coli (E. coli) / References: UniProt: P00579 |
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#6: Protein | Mass: 8903.552 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Gene: narL, frdR, b1221, JW1212 / Production host: Escherichia coli (E. coli) / References: UniProt: P0AF28 |
-DNA chain , 2 types, 2 molecules 12
#7: DNA chain | Mass: 21307.693 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli) |
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#8: DNA chain | Mass: 21164.541 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli) |
-RNA chain , 1 types, 1 molecules 3
#9: RNA chain | Mass: 1118.619 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
#10: Chemical | #11: Chemical | ChemComp-MG / | |
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-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: E. coli NarL transcription activation complex on the yeaR promoter Type: COMPLEX / Entity ID: #1-#9 / Source: RECOMBINANT |
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Molecular weight | Value: 0.625 MDa / Experimental value: YES |
Source (natural) | Organism: Escherichia coli (E. coli) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 8 |
Specimen | Conc.: 15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 290 K |
-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 magnification: 130000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (min): 77 K |
Image recording | Average exposure time: 2 sec. / Electron dose: 42 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8727 |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
Image scans | Width: 5769 / Height: 4092 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 2554259 | ||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 104500 / Algorithm: SIMULTANEOUS ITERATIVE (SIRT) / Symmetry type: POINT | ||||||||||||||||||||||||||||||||
Atomic model building | Protocol: OTHER / Space: REAL | ||||||||||||||||||||||||||||||||
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