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Yorodumi- PDB-7rdq: Cryo-EM structure of Thermus thermophilus reiterative transcripti... -
+Open data
-Basic information
Entry | Database: PDB / ID: 7rdq | ||||||
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Title | Cryo-EM structure of Thermus thermophilus reiterative transcription complex with 11nt oligo-G RNA | ||||||
Components |
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Keywords | TRANSCRIPTION / Thermus thermophilus / transcription initiation | ||||||
Function / homology | Function and homology information sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / DNA-templated transcription / magnesium ion binding / DNA binding ...sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / 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 / metal ion binding / cytoplasm Similarity search - Function | ||||||
Biological species | Thermus thermophilus HB8 (bacteria) synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å | ||||||
Authors | Liu, Y. / Ebright, R.H. | ||||||
Funding support | United States, 1items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2022 Title: Structural and mechanistic basis of reiterative transcription initiation. Authors: Yu Liu / Libing Yu / Chirangini Pukhrambam / Jared T Winkelman / Emre Firlar / Jason T Kaelber / Yu Zhang / Bryce E Nickels / Richard H Ebright / Abstract: Reiterative transcription initiation, observed at promoters that contain homopolymeric sequences at the transcription start site, generates RNA products having 5' sequences noncomplementary to the ...Reiterative transcription initiation, observed at promoters that contain homopolymeric sequences at the transcription start site, generates RNA products having 5' sequences noncomplementary to the DNA template. Here, using crystallography and cryoelectron microscopy to define structures, protein-DNA photocrosslinking to map positions of RNAP leading and trailing edges relative to DNA, and single-molecule DNA nanomanipulation to assess RNA polymerase (RNAP)-dependent DNA unwinding, we show that RNA extension in reiterative transcription initiation 1) occurs without DNA scrunching; 2) involves a short, 2- to 3-bp, RNA-DNA hybrid; and 3) generates RNA that exits RNAP through the portal by which scrunched nontemplate-strand DNA exits RNAP in standard transcription initiation. The results establish that, whereas RNA extension in standard transcription initiation proceeds through a scrunching mechanism, RNA extension in reiterative transcription initiation proceeds through a slippage mechanism, with slipping of RNA relative to DNA within a short RNA-DNA hybrid, and with extrusion of RNA from RNAP through an alternative RNA exit. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7rdq.cif.gz | 647.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7rdq.ent.gz | 507.4 KB | Display | PDB format |
PDBx/mmJSON format | 7rdq.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7rdq_validation.pdf.gz | 1 MB | Display | wwPDB validaton report |
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Full document | 7rdq_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 7rdq_validation.xml.gz | 90.4 KB | Display | |
Data in CIF | 7rdq_validation.cif.gz | 141.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/rd/7rdq ftp://data.pdbj.org/pub/pdb/validation_reports/rd/7rdq | HTTPS FTP |
-Related structure data
Related structure data | 24424MC 7mlbC 7mliC 7mljC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data |
-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: 35056.164 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / Strain: HB8 / ATCC 27634 / DSM 579 / References: UniProt: Q5SHR6, DNA-directed RNA polymerase #2: Protein | | Mass: 125436.539 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / Strain: HB8 / ATCC 27634 / DSM 579 / References: UniProt: Q8RQE9, DNA-directed RNA polymerase #3: Protein | | Mass: 170997.391 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / Strain: HB8 / ATCC 27634 / DSM 579 / References: UniProt: Q8RQE8, DNA-directed RNA polymerase #4: Protein | | Mass: 11533.316 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / Strain: HB8 / ATCC 27634 / DSM 579 / References: UniProt: Q8RQE7, DNA-directed RNA polymerase |
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-DNA (31-MER) ... , 2 types, 2 molecules HG
#6: DNA chain | Mass: 10331.629 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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#7: DNA chain | Mass: 10100.500 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-Protein / RNA chain , 2 types, 2 molecules FI
#5: Protein | Mass: 48598.031 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Thermus thermophilus HB8 (bacteria) / Strain: HB8 / ATCC 27634 / DSM 579 / Gene: sigA, TTHA0532 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q5SKW1 |
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#8: RNA chain | Mass: 3752.306 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-Non-polymers , 2 types, 7 molecules
#9: Chemical | ChemComp-MG / #10: Chemical | |
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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 | Name: reiterative transcription initiation complex with 11nt oligo-G RNA Type: COMPLEX / Entity ID: #1-#8 / Source: MULTIPLE SOURCES | ||||||||||||||||||||
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Molecular weight | Value: 0.425 MDa / Experimental value: NO | ||||||||||||||||||||
Source (natural) | Organism: Thermus thermophilus HB8 (bacteria) | ||||||||||||||||||||
Buffer solution | pH: 7.9 | ||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.69 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 R2/1 | ||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K |
-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: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: -2800 nm / Nominal defocus min: -1200 nm |
Image recording | Average exposure time: 0.2 sec. / Electron dose: 0.994 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3424 |
Image scans | Movie frames/image: 30 / Used frames/image: 1-30 |
-Processing
Software |
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 2143284 | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 339426 / Num. of class averages: 3 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building |
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Atomic model building | Accession code: 4G7H / Initial refinement model-ID: 1 / PDB-ID: 4G7H / Source name: PDB / Type: experimental model
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Refinement | Cross valid method: NONE |