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Yorodumi- PDB-5ms0: pseudo-atomic model of the RNA polymerase lambda-based antitermin... -
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-Basic information
Entry | Database: PDB / ID: 5ms0 | ||||||
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Title | pseudo-atomic model of the RNA polymerase lambda-based antitermination complex solved by cryo-EM | ||||||
Components |
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Keywords | TRANSCRIPTION/DNA/RNA / DNA-DEPENDENT RNA POLYMERASE / BACTERIAL TRANSCRIPTION / TERNARY ELONGATION COMPLEX / ANTITERMINATION / TRANSCRIPTION-DNA-RNA complex | ||||||
Function / homology | Function and homology information bacterial-type RNA polymerase core enzyme binding / RNA polymerase binding / transcription elongation-coupled chromatin remodeling / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly / transcription antitermination factor activity, RNA binding ...bacterial-type RNA polymerase core enzyme binding / RNA polymerase binding / transcription elongation-coupled chromatin remodeling / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly / transcription antitermination factor activity, RNA binding / bacterial-type flagellum-dependent cell motility / nitrate assimilation / transcription elongation factor complex / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription initiation / cell motility / DNA-templated transcription termination / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / RNA stem-loop binding / ribosome biogenesis / protein complex oligomerization / small ribosomal subunit / response to heat / cytosolic small ribosomal subunit / protein-containing complex assembly / intracellular iron ion homeostasis / cytoplasmic translation / tRNA binding / single-stranded RNA binding / protein dimerization activity / structural constituent of ribosome / protein domain specific binding / DNA-binding transcription factor activity / response to antibiotic / nucleotide binding / regulation of transcription by RNA polymerase II / magnesium ion binding / DNA binding / RNA binding / zinc ion binding / membrane / cytoplasm / cytosol Similarity search - Function | ||||||
Biological species | Escherichia phage lambda (virus) Escherichia coli (E. coli) Escherichia coli K-12 (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 9.8 Å | ||||||
Authors | Said, N. / Krupp, F. | ||||||
Funding support | Germany, 1items
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Citation | Journal: Nat Microbiol / Year: 2017 Title: Structural basis for λN-dependent processive transcription antitermination. Authors: Nelly Said / Ferdinand Krupp / Ekaterina Anedchenko / Karine F Santos / Olexandr Dybkov / Yong-Heng Huang / Chung-Tien Lee / Bernhard Loll / Elmar Behrmann / Jörg Bürger / Thorsten Mielke ...Authors: Nelly Said / Ferdinand Krupp / Ekaterina Anedchenko / Karine F Santos / Olexandr Dybkov / Yong-Heng Huang / Chung-Tien Lee / Bernhard Loll / Elmar Behrmann / Jörg Bürger / Thorsten Mielke / Justus Loerke / Henning Urlaub / Christian M T Spahn / Gert Weber / Markus C Wahl / Abstract: λN-mediated processive antitermination constitutes a paradigmatic transcription regulatory event, during which phage protein λN, host factors NusA, NusB, NusE and NusG, and an RNA nut site render ...λN-mediated processive antitermination constitutes a paradigmatic transcription regulatory event, during which phage protein λN, host factors NusA, NusB, NusE and NusG, and an RNA nut site render elongating RNA polymerase termination-resistant. The structural basis of the process has so far remained elusive. Here we describe a crystal structure of a λN-NusA-NusB-NusE-nut site complex and an electron cryo-microscopic structure of a complete transcription antitermination complex, comprising RNA polymerase, DNA, nut site RNA, all Nus factors and λN, validated by crosslinking/mass spectrometry. Due to intrinsic disorder, λN can act as a multiprotein/RNA interaction hub, which, together with nut site RNA, arranges NusA, NusB and NusE into a triangular complex. This complex docks via the NusA N-terminal domain and the λN C-terminus next to the RNA exit channel on RNA polymerase. Based on the structures, comparative crosslinking analyses and structure-guided mutagenesis, we hypothesize that λN mounts a multipronged strategy to reprogram the transcriptional machinery, which may include (1) the λN C terminus clamping the RNA exit channel, thus stabilizing the DNA:RNA hybrid; (2) repositioning of NusA and RNAP elements, thus redirecting nascent RNA and sequestering the upstream branch of a terminator hairpin; and (3) hindering RNA engagement of termination factor ρ and/or obstructing ρ translocation on the transcript. | ||||||
History |
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-Structure visualization
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Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
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PDBx/mmCIF format | 5ms0.cif.gz | 651.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb5ms0.ent.gz | 429.6 KB | Display | PDB format |
PDBx/mmJSON format | 5ms0.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 5ms0_validation.pdf.gz | 806.5 KB | Display | wwPDB validaton report |
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Full document | 5ms0_full_validation.pdf.gz | 822.7 KB | Display | |
Data in XML | 5ms0_validation.xml.gz | 87.5 KB | Display | |
Data in CIF | 5ms0_validation.cif.gz | 148 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ms/5ms0 ftp://data.pdbj.org/pub/pdb/validation_reports/ms/5ms0 | HTTPS FTP |
-Related structure data
Related structure data | 3561MC 5lm7C 5lm9C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Protein , 3 types, 3 molecules NEL
#1: Protein | Mass: 9877.343 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Details: lambda N factor / Source: (gene. exp.) Escherichia phage lambda (virus) / Gene: N, lambdap49 / Production host: Escherichia coli (E. coli) / References: UniProt: P03045 |
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#6: Protein | Mass: 11340.067 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: rpsJ, nusE, b3321, JW3283 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A7R5 |
#11: Protein | Mass: 15709.967 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: nusB, groNB, ssyB, b0416, JW0406 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A780 |
-RNA chain , 2 types, 2 molecules RH
#2: RNA chain | Mass: 9339.650 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) |
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#8: RNA chain | Mass: 4492.788 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) |
-DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules ABCDO
#3: 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 #4: Protein | | Mass: 150804.922 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 (E. coli) / References: UniProt: P0A8V2, DNA-directed RNA polymerase #5: Protein | | Mass: 156537.031 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: rpoC, tabB, b3988, JW3951 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A8T7, DNA-directed RNA polymerase #13: 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 (E. coli) / References: UniProt: P0A800, DNA-directed RNA polymerase |
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-Transcription termination/antitermination protein ... , 2 types, 2 molecules FM
#7: Protein | Mass: 20688.654 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: nusG, b3982, JW3945 / Production host: Escherichia coli (E. coli) / References: UniProt: P0AFG0 |
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#12: Protein | Mass: 55059.828 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: nusA, b3169, JW3138 / Production host: Escherichia coli (E. coli) / References: UniProt: P0AFF6 |
-DNA chain , 2 types, 2 molecules IJ
#9: DNA chain | Mass: 8355.408 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) |
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#10: DNA chain | Mass: 11942.696 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) |
-Non-polymers , 2 types, 3 molecules
#14: Chemical | ChemComp-MG / |
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#15: Chemical |
-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: lambdaN-dependent antitermination complex / Type: COMPLEX / Entity ID: #1-#13 / Source: RECOMBINANT |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 10 mM HEPES-NaOH, pH 7.5, 50 mM NaCl, 1 mM DTT |
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Tecnai Polara / Image courtesy: FEI Company |
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Microscopy | Model: FEI POLARA 300 |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 1 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
-Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION |
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Symmetry | Point symmetry: C1 (asymmetric) |
3D reconstruction | Resolution: 9.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 23983 / Symmetry type: POINT |