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Entry
Database: PDB / ID: 5ms0
Titlepseudo-atomic model of the RNA polymerase lambda-based antitermination complex solved by cryo-EM
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • (Transcription termination/antitermination protein ...) x 2
  • 30S ribosomal protein S10
  • Antitermination protein N
  • DNAI
  • DNAII
  • RNA transcription bubble
  • Transcription antitermination protein NusB
  • nascent RNA
KeywordsTRANSCRIPTION/DNA/RNA / DNA-DEPENDENT RNA POLYMERASE / BACTERIAL TRANSCRIPTION / TERNARY ELONGATION COMPLEX / ANTITERMINATION / TRANSCRIPTION-DNA-RNA complex
Function / homology
Function and homology information


RNA polymerase binding / RNA polymerase complex / submerged biofilm formation / transcription elongation-coupled chromatin remodeling / cellular response to cell envelope stress / regulation of DNA-templated transcription initiation / protein complex oligomerization / bacterial-type flagellum assembly / transcription antitermination factor activity, RNA binding / bacterial-type RNA polymerase core enzyme binding ...RNA polymerase binding / RNA polymerase complex / submerged biofilm formation / transcription elongation-coupled chromatin remodeling / cellular response to cell envelope stress / regulation of DNA-templated transcription initiation / protein complex oligomerization / bacterial-type flagellum assembly / transcription antitermination factor activity, RNA binding / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / bacterial-type flagellum-dependent cell motility / nitrate assimilation / regulation of DNA-templated transcription elongation / transcription elongation factor complex / transcription antitermination / cell motility / DNA-templated transcription initiation / DNA-templated transcription termination / ribonucleoside binding / RNA stem-loop binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / ribosome biogenesis / response to heat / small ribosomal subunit / protein-containing complex assembly / cytosolic small ribosomal subunit / intracellular iron ion homeostasis / cytoplasmic translation / tRNA binding / single-stranded RNA binding / protein dimerization activity / structural constituent of ribosome / DNA-binding transcription factor activity / protein domain specific binding / response to antibiotic / nucleotide binding / regulation of transcription by RNA polymerase II / magnesium ion binding / DNA binding / RNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
Antitermination protein N, arginine-rich motif / 36-mer N-terminal peptide of the N protein (N36) / NusB antitermination factor / NusB/RsmB/TIM44 / NusB family / NusB-like superfamily / Transcription termination factor NusA, C-terminal duplication / Transcription termination factor NusA / Transcription factor NusA, N-terminal / KH domain, NusA-like ...Antitermination protein N, arginine-rich motif / 36-mer N-terminal peptide of the N protein (N36) / NusB antitermination factor / NusB/RsmB/TIM44 / NusB family / NusB-like superfamily / Transcription termination factor NusA, C-terminal duplication / Transcription termination factor NusA / Transcription factor NusA, N-terminal / KH domain, NusA-like / NusA, N-terminal domain superfamily / NusA N-terminal domain / NusA-like first KH domain / Transcription termination/antitermination protein NusA, bacterial / : / Transcription antitermination protein, NusG / Transcription antitermination protein, NusG, bacteria, conserved site / Transcription termination factor nusG signature. / NusG-like / Transcription termination factor nusG / NusG, N-terminal / In Spt5p, this domain may confer affinity for Spt4p. It possesses a RNP-like fold. / Type-1 KH domain profile. / DNA repair Rad51/transcription factor NusA, alpha-helical / NusG, N-terminal domain superfamily / Helix-hairpin-helix domain / S1 domain profile. / DNA-directed RNA polymerase, omega subunit / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase beta subunit external 1 domain / RNA polymerase, alpha subunit, C-terminal / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase beta subunit, bacterial-type / DNA-directed RNA polymerase, alpha subunit / Ribosomal protein S1-like RNA-binding domain / S1 RNA binding domain / S1 domain / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, N-terminal / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb2, domain 2 / RNA polymerase I subunit A N-terminus / RNA polymerase, RBP11-like subunit / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, OB-fold / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerases beta chain signature. / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 6 / Ribosomal protein S10, conserved site / Ribosomal protein S10 signature. / Ribosomal protein S10 / K homology domain superfamily, prokaryotic type / K homology domain-like, alpha/beta / Ribosomal protein S10p/S20e / Ribosomal protein S10 domain / Ribosomal protein S10 domain superfamily / Ribosomal protein S10p/S20e / KOW (Kyprides, Ouzounis, Woese) motif. / Translation protein SH3-like domain superfamily / KOW motif / KOW / Ribosomal protein L2, domain 2 / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / Antitermination protein N / Transcription antitermination protein NusB / Small ribosomal subunit protein uS10 / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' ...DNA / DNA (> 10) / RNA / RNA (> 10) / Antitermination protein N / Transcription antitermination protein NusB / Small ribosomal subunit protein uS10 / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / Transcription termination/antitermination protein NusA / Transcription termination/antitermination protein NusG
Similarity search - Component
Biological speciesEscherichia phage lambda (virus)
Escherichia coli (E. coli)
Escherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 9.8 Å
AuthorsSaid, N. / Krupp, F.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research FoundationSFB740 Germany
CitationJournal: 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
DepositionDec 29, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 3, 2017Provider: repository / Type: Initial release
Revision 1.1May 10, 2017Group: Database references
Revision 1.2Oct 31, 2018Group: Author supporting evidence / Data collection ...Author supporting evidence / Data collection / Database references / Source and taxonomy / Structure summary
Category: citation_author / em_image_scans ...citation_author / em_image_scans / entity / entity_name_com / entity_src_gen / pdbx_audit_support / struct_ref / struct_ref_seq / struct_ref_seq_dif
Item: _citation_author.identifier_ORCID / _entity.pdbx_description ..._citation_author.identifier_ORCID / _entity.pdbx_description / _entity_src_gen.gene_src_common_name / _entity_src_gen.pdbx_gene_src_gene / _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id / _entity_src_gen.pdbx_gene_src_scientific_name / _pdbx_audit_support.funding_organization / _struct_ref.db_code / _struct_ref.pdbx_db_accession / _struct_ref_seq.pdbx_db_accession / _struct_ref_seq_dif.pdbx_seq_db_accession_code
Revision 1.3May 15, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
N: Antitermination protein N
R: nascent RNA
A: DNA-directed RNA polymerase subunit alpha
B: DNA-directed RNA polymerase subunit alpha
C: DNA-directed RNA polymerase subunit beta
D: DNA-directed RNA polymerase subunit beta'
E: 30S ribosomal protein S10
F: Transcription termination/antitermination protein NusG
H: RNA transcription bubble
I: DNAI
J: DNAII
L: Transcription antitermination protein NusB
M: Transcription termination/antitermination protein NusA
O: DNA-directed RNA polymerase subunit omega
hetero molecules


Theoretical massNumber of molelcules
Total (without water)537,67017
Polymers537,51514
Non-polymers1553
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 3 molecules NEL

#1: Protein Antitermination protein N / Regulatory protein N / PN


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
#6: Protein 30S ribosomal protein S10 / Small ribosomal subunit protein uS10


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 Transcription antitermination protein NusB / Antitermination factor NusB / N utilization substance protein B


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

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RNA chain , 2 types, 2 molecules RH

#2: RNA chain nascent RNA


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)
#8: RNA chain RNA transcription bubble


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)

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DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules ABCDO

#3: Protein DNA-directed RNA polymerase subunit alpha / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


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 DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


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 DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


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 DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


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 Transcription termination/antitermination protein NusG


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
#12: Protein Transcription termination/antitermination protein NusA / N utilization substance protein A / Transcription termination/antitermination L factor


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

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DNA chain , 2 types, 2 molecules IJ

#9: DNA chain DNAI


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)
#10: DNA chain DNAII


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)

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Non-polymers , 2 types, 3 molecules

#14: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#15: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: lambdaN-dependent antitermination complex / Type: COMPLEX / Entity ID: #1-#13 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding 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
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 1 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 9.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 23983 / Symmetry type: POINT

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