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- PDB-8oki: Cryo-EM structure of Pyrococcus furiosus transcription elongation... -
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Basic information
Entry | Database: PDB / ID: 8oki | ||||||
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Title | Cryo-EM structure of Pyrococcus furiosus transcription elongation complex bound to Spt4/5 | ||||||
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![]() | TRANSCRIPTION / RNA / Polymerase / Elongation complex / Pyrococcus furiosus / DNA / Archaea | ||||||
Function / homology | ![]() transcription elongation-coupled chromatin remodeling / RNA polymerase III activity / transcription initiation at RNA polymerase III promoter / RNA polymerase II activity / tRNA transcription by RNA polymerase III / RNA polymerase I activity / translation elongation factor activity / DNA-directed RNA polymerase complex / regulation of DNA-templated transcription elongation / DNA-templated transcription initiation ...transcription elongation-coupled chromatin remodeling / RNA polymerase III activity / transcription initiation at RNA polymerase III promoter / RNA polymerase II activity / tRNA transcription by RNA polymerase III / RNA polymerase I activity / translation elongation factor activity / DNA-directed RNA polymerase complex / regulation of DNA-templated transcription elongation / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / ribosome / structural constituent of ribosome / DNA-templated transcription / mRNA binding / nucleotide binding / regulation of transcription by RNA polymerase II / regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm Similarity search - Function | ||||||
Biological species | ![]() ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.45 Å | ||||||
![]() | Tarau, D.M. / Reichelt, R. / Heiss, F.B. / Pilsl, M. / Hausner, W. / Engel, C. / Grohmann, D. | ||||||
Funding support | ![]()
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![]() | ![]() Title: Structural basis of archaeal RNA polymerase transcription elongation and Spt4/5 recruitment. Authors: Daniela Tarău / Felix Grünberger / Michael Pilsl / Robert Reichelt / Florian Heiß / Sabine König / Henning Urlaub / Winfried Hausner / Christoph Engel / Dina Grohmann / ![]() Abstract: Archaeal transcription is carried out by a multi-subunit RNA polymerase (RNAP) that is highly homologous in structure and function to eukaryotic RNAP II. Among the set of basal transcription factors, ...Archaeal transcription is carried out by a multi-subunit RNA polymerase (RNAP) that is highly homologous in structure and function to eukaryotic RNAP II. Among the set of basal transcription factors, only Spt5 is found in all domains of life, but Spt5 has been shaped during evolution, which is also reflected in the heterodimerization of Spt5 with Spt4 in Archaea and Eukaryotes. To unravel the mechanistic basis of Spt4/5 function in Archaea, we performed structure-function analyses using the archaeal transcriptional machinery of Pyrococcus furiosus (Pfu). We report single-particle cryo-electron microscopy reconstructions of apo RNAP and the archaeal elongation complex (EC) in the absence and presence of Spt4/5. Surprisingly, Pfu Spt4/5 also binds the RNAP in the absence of nucleic acids in a distinct super-contracted conformation. We show that the RNAP clamp/stalk module exhibits conformational flexibility in the apo state of RNAP and that the enzyme contracts upon EC formation or Spt4/5 engagement. We furthermore identified a contact of the Spt5-NGN domain with the DNA duplex that stabilizes the upstream boundary of the transcription bubble and impacts Spt4/5 activity in vitro. This study, therefore, provides the structural basis for Spt4/5 function in archaeal transcription and reveals a potential role beyond the well-described support of elongation. | ||||||
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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PDBx/mmCIF format | ![]() | 648 KB | Display | ![]() |
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PDB format | ![]() | 509.4 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 16929MC ![]() 8croC ![]() 8orqC ![]() 8p2iC ![]() 8rboC 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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Components
-DNA-directed RNA polymerase subunit ... , 11 types, 11 molecules ABCDHKLNPEF
#1: Protein | Mass: 103612.250 Da / Num. of mol.: 1 / Mutation: 0 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() References: UniProt: A0A5C0XPL7, DNA-directed RNA polymerase |
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#2: Protein | Mass: 127188.898 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
#3: Protein | Mass: 44468.156 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
#4: Protein | Mass: 31688.195 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
#6: Protein | Mass: 9260.792 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
#8: Protein | Mass: 6243.490 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
#9: Protein | Mass: 11132.764 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
#10: Protein | Mass: 7800.158 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
#11: Protein/peptide | Mass: 5770.050 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
#15: Protein | Mass: 21731.455 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
#16: Protein | Mass: 14116.311 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
-Transcription elongation factor ... , 2 types, 2 molecules GI
#5: Protein | Mass: 16816.600 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
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#7: Protein | Mass: 6965.045 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
-DNA chain , 2 types, 2 molecules XY
#12: DNA chain | Mass: 8043.188 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() |
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#13: DNA chain | Mass: 7932.130 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() |
-RNA chain , 1 types, 1 molecules Z
#14: RNA chain | Mass: 2909.823 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() |
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-Non-polymers , 2 types, 7 molecules ![](data/chem/img/ZN.gif)
![](data/chem/img/MG.gif)
![](data/chem/img/MG.gif)
#17: Chemical | ChemComp-ZN / #18: 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 |
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Sample preparation
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Buffer solution | pH: 8 | ||||||||||||||||||||||||||||||
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||
Specimen support | Grid material: GOLD / Grid type: UltrAuFoil R1.2/1.3 | ||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K |
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Electron microscopy imaging
Microscopy | Model: JEOL CRYO ARM 200 |
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Electron gun | Electron source: ![]() |
Electron lens | Mode: OTHER / Nominal defocus max: 22000 nm / Nominal defocus min: 1000 nm |
Image recording | Electron dose: 40 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
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Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
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3D reconstruction | Resolution: 3.45 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 52527 / Symmetry type: POINT | ||||||||||||||||||||||||
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