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- PDB-8ehf: Cryo-EM structure of his-elemental paused elongation complex with... -
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Open data
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Basic information
Entry | Database: PDB / ID: 8ehf | |||||||||
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Title | Cryo-EM structure of his-elemental paused elongation complex with an unfolded TL (1) | |||||||||
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Function / homology | ![]() ![]() ![]() ![]() ![]() ![]() ![]() Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() | |||||||||
Method | ![]() ![]() ![]() | |||||||||
![]() | Kang, J.Y. / Chen, J. / Llewellyn, E. / Landick, R. / Darst, S.A. | |||||||||
Funding support | ![]()
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![]() | ![]() Title: An ensemble of interconverting conformations of the elemental paused transcription complex creates regulatory options. Authors: Jin Young Kang / Tatiana V Mishanina / Yu Bao / James Chen / Eliza Llewellyn / James Liu / Seth A Darst / Robert Landick / ![]() ![]() Abstract: Transcriptional pausing underpins the regulation of cellular RNA synthesis, but its mechanism remains incompletely understood. Sequence-specific interactions of DNA and RNA with the dynamic, ...Transcriptional pausing underpins the regulation of cellular RNA synthesis, but its mechanism remains incompletely understood. Sequence-specific interactions of DNA and RNA with the dynamic, multidomain RNA polymerase (RNAP) trigger reversible conformational changes at pause sites that temporarily interrupt the nucleotide addition cycle. These interactions initially rearrange the elongation complex (EC) into an elemental paused EC (ePEC). ePECs can form longer-lived PECs by further rearrangements or interactions of diffusible regulators. For both bacterial and mammalian RNAPs, a half-translocated state in which the next DNA template base fails to load into the active site appears central to the ePEC. Some RNAPs also swivel interconnected modules that may stabilize the ePEC. However, it is unclear whether swiveling and half-translocation are requisite features of a single ePEC state or if multiple ePEC states exist. Here, we use cryo-electron microscopy (cryo-EM) analysis of ePECs with different RNA-DNA sequences combined with biochemical probes of ePEC structure to define an interconverting ensemble of ePEC states. ePECs occupy either pre- or half-translocated states but do not always swivel, indicating that difficulty in forming the posttranslocated state at certain RNA-DNA sequences may be the essence of the ePEC. The existence of multiple ePEC conformations has broad implications for transcriptional regulation. | |||||||||
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 585.4 KB | Display | ![]() |
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PDB format | ![]() | 470.2 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 28146MC ![]() 8eg7C ![]() 8eg8C ![]() 8egbC ![]() 8eh8C ![]() 8eh9C ![]() 8ehaC ![]() 8ehiC C: citing same article ( M: map data used to model this data |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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1 |
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Components
-DNA chain , 2 types, 2 molecules AB
#1: DNA chain | Mass: 9881.364 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() ![]() |
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#2: DNA chain | Mass: 9706.256 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() ![]() |
-RNA chain , 1 types, 1 molecules R
#3: RNA chain | ![]() Mass: 6047.593 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() ![]() |
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-DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules GHIJK
#4: Protein | ![]() Mass: 26459.125 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() ![]() ![]() ![]() #5: Protein | | ![]() Mass: 150820.875 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() ![]() ![]() ![]() #6: Protein | | ![]() Mass: 155334.719 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() ![]() ![]() ![]() #7: Protein | | ![]() Mass: 10249.547 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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-Non-polymers , 3 types, 4 molecules ![](data/chem/img/4QM.gif)
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#8: Chemical | ChemComp-4QM / ( |
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#9: Chemical | ChemComp-MG / |
#10: Chemical |
-Details
Has ligand of interest | N |
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-Experimental details
-Experiment
Experiment | Method: ![]() |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: ![]() |
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Sample preparation
Component | Name: his-elemental paused elongation complex / Type: COMPLEX Details: An RNA polymerase elongation complex was assembled at the weak elemental pausing site derived from his-pause sequence. Entity ID: #1-#7 / Source: MULTIPLE SOURCES | |||||||||||||||||||||||||
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Molecular weight | Value: 0.45 MDa / Experimental value: NO | |||||||||||||||||||||||||
Source (natural) | Organism: ![]() ![]() ![]() | |||||||||||||||||||||||||
Source (recombinant) | Organism: ![]() ![]() ![]() | |||||||||||||||||||||||||
Buffer solution | pH: 8 | |||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied![]() ![]() | |||||||||||||||||||||||||
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 % / Chamber temperature: 295 K / Details: blot force 0, blot time 3.5 s |
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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![]() ![]() |
Electron lens | Mode: BRIGHT FIELD![]() ![]() |
Specimen holder | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 10 sec. / Electron dose: 47.34 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6939 |
Image scans | Width: 3710 / Height: 3838 / Movie frames/image: 50 / Used frames/image: 1-50 |
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Processing
Software | Name: PHENIX / Version: 1.19.2_4158: / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction![]() | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 2126200 | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry![]() | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction![]() | Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 432300 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: REAL | ||||||||||||||||||||||||||||||||||||||||
Atomic model building |
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Refine LS restraints |
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