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- PDB-6gov: Structure of THE RNA POLYMERASE LAMBDA-BASED ANTITERMINATION COMPLEX -

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Entry
Database: PDB / ID: 6gov
TitleStructure of THE RNA POLYMERASE LAMBDA-BASED ANTITERMINATION COMPLEX
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • (Transcription termination/antitermination protein ...) x 2
  • 30S ribosomal protein S10
  • Antitermination protein N
  • DNA (I)
  • DNA (II)
  • RNA (TRANSCRIPTION BUBBLE)
  • Transcription antitermination protein NusB
KeywordsTRANSCRIPTION / TRANSCRIPTION/DNA/RNA / DNA-DEPENDENT RNA POLYMERASE / BACTERIAL TRANSCRIPTION / TERNARY ELONGATION COMPLEX / ANTITERMINATION / TRANSCRIPTION-DNA-RNA COMPLEX / transcription
Function / homologyHelix-hairpin-helix domain / NusB-like superfamily / Ribosomal protein S10p/S20e / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / Ribosomal protein S10 domain superfamily / NusG, N-terminal domain superfamily / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit ...Helix-hairpin-helix domain / NusB-like superfamily / Ribosomal protein S10p/S20e / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / Ribosomal protein S10 domain superfamily / NusG, N-terminal domain superfamily / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / NusA, N-terminal domain superfamily / RPB6/omega subunit-like superfamily / Transcription termination/antitermination protein NusA, bacterial / RNA polymerase Rpb2, domain 6 / Ribosomal protein S10 domain / KH domain, NusA-like / RNA-binding domain, S1 / Antitermination protein N, arginine-rich motif / DNA-directed RNA polymerase, beta subunit, external 1 domain / Ribosomal protein S10, conserved site / K homology domain-like, alpha/beta / Transcription antitermination protein, NusG, bacteria, conserved site / RNA polymerase Rpb2, OB-fold / Ribosomal protein L2, domain 2 / KOW motif / S1 RNA binding domain / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 5 / S1 domain profile. / Type-1 KH domain profile. / RNA polymerases beta chain signature. / Transcription termination factor nusG signature. / Ribosomal protein S10 signature. / NusA-like KH domain / 36-mer N-terminal peptide of the N protein (N36) / RNA polymerase beta subunit external 1 domain / NusA N-terminal domain / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 3 / RNA polymerase beta subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 7 / Bacterial RNA polymerase, alpha chain C terminal domain / Transcription termination factor nusG / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase Rpb6 / NusB family / RNA polymerase Rpb3/RpoA insert domain / Transcription factor NusA, N-terminal / DNA-directed RNA polymerase, subunit 2 / Nucleic acid-binding, OB-fold / RNA polymerase, subunit omega/K/RPB6 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 3 / NusG, N-terminal / RNA polymerase, N-terminal / NusB/RsmB/TIM44 / RNA polymerase, beta subunit, conserved site / KOW / DNA-directed RNA polymerase, omega subunit / S1 domain / RNA polymerase subunit, RPB6/omega / Transcription antitermination protein, NusG / RNA polymerase, alpha subunit / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / Ribosomal protein S10 / RNA polymerase Rpb2, domain 7 / DNA-directed RNA polymerase, alpha subunit / RNA polymerase, beta subunit, protrusion / RNA polymerase Rpb2, domain 3 / Translation protein SH3-like domain superfamily / K homology domain superfamily, prokaryotic type / Transcription termination factor NusA / Transcription termination factor NusA, C-terminal duplication / DNA-directed RNA polymerase beta subunit, bacterial-type / DNA repair Rad51/transcription factor NusA, alpha-helical / RNA polymerase, alpha subunit, C-terminal / DNA-directed RNA polymerase, insert domain / RNA polymerase Rpb2, domain 2 / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / NusB antitermination factor / DNA-templated transcription, elongation / bacterial-type RNA polymerase core enzyme binding / regulation of DNA-templated transcription, elongation / RNA stem-loop binding / transcription antitermination factor activity, RNA binding / transcription antitermination / ribonucleoside binding / DNA-templated transcription, termination / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / tRNA binding / ribosome / structural constituent of ribosome / translation
Function and homology information
Specimen sourceEscherichia coli O157:H7 (bacteria)
Escherichia phage lambda (bacteriophage)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.7 Å resolution
AuthorsLoll, B. / Krupp, F. / Said, N. / Huang, Y. / Buerger, J. / Mielke, T. / Spahn, C.M.T. / Wahl, M.C.
CitationJournal: Mol. Cell / Year: 2019
Title: Structural Basis for the Action of an All-Purpose Transcription Anti-termination Factor.
Authors: Ferdinand Krupp / Nelly Said / Yong-Heng Huang / Bernhard Loll / Jörg Bürger / Thorsten Mielke / Christian M T Spahn / Markus C Wahl
Abstract: Bacteriophage λN protein, a model anti-termination factor, binds nascent RNA and host Nus factors, rendering RNA polymerase resistant to all pause and termination signals. A 3.7-Å-resolution ...Bacteriophage λN protein, a model anti-termination factor, binds nascent RNA and host Nus factors, rendering RNA polymerase resistant to all pause and termination signals. A 3.7-Å-resolution cryo-electron microscopy structure and structure-informed functional analyses reveal a multi-pronged strategy by which the intrinsically unstructured λN directly modifies RNA polymerase interactions with the nucleic acids and subverts essential functions of NusA, NusE, and NusG to reprogram the transcriptional apparatus. λN repositions NusA and remodels the β subunit flap tip, which likely precludes folding of pause or termination RNA hairpins in the exit tunnel and disrupts termination-supporting interactions of the α subunit C-terminal domains. λN invades and traverses the RNA polymerase hybrid cavity, likely stabilizing the hybrid and impeding pause- or termination-related conformational changes of polymerase. λN also lines upstream DNA, seemingly reinforcing anti-backtracking and anti-swiveling by NusG. Moreover, λN-repositioned NusA and NusE sequester the NusG C-terminal domain, counteracting ρ-dependent termination. Other anti-terminators likely utilize similar mechanisms to enable processive transcription.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jun 4, 2018 / Release: Feb 13, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0Feb 13, 2019Structure modelrepositoryInitial release
1.1Feb 27, 2019Structure modelData collection / Database referencescitation / pdbx_database_proc / pdbx_seq_map_depositor_info_citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _pdbx_seq_map_depositor_info.one_letter_code_mod
1.2Mar 6, 2019Structure modelData collection / Database referencescitation / citation_author / pdbx_database_proc / pdbx_seq_map_depositor_info_citation.journal_abbrev / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name / _pdbx_seq_map_depositor_info.one_letter_code_mod

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Assembly

Deposited unit
A: Transcription termination/antitermination protein NusA
B: Transcription antitermination protein NusB
E: 30S ribosomal protein S10
G: Transcription termination/antitermination protein NusG
N: Antitermination protein N
U: DNA-directed RNA polymerase subunit alpha
V: DNA-directed RNA polymerase subunit alpha
W: DNA-directed RNA polymerase subunit omega
X: DNA-directed RNA polymerase subunit beta
Y: DNA-directed RNA polymerase subunit beta'
K: DNA (I)
L: DNA (II)
R: RNA (TRANSCRIPTION BUBBLE)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)568,53516
Polyers568,38013
Non-polymers1553
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
MethodPISA

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Components

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Transcription termination/antitermination protein ... , 2 types, 2 molecules AG

#1: Protein/peptide Transcription termination/antitermination protein NusA


Mass: 55030.789 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli O157:H7 (bacteria)
Gene: nusA, Z4530, ECs4050Newcastle University Students' Association
Production host: Escherichia coli (E. coli) / References: UniProt: P0AFF8
#4: Protein/peptide Transcription termination/antitermination protein NusG


Mass: 20831.838 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli O157:H7 (bacteria) / Gene: nusG, Z5555, ECs4905 / Production host: Escherichia coli (E. coli) / References: UniProt: P0AFG1

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Protein/peptide , 3 types, 3 molecules BEN

#2: Protein/peptide Transcription antitermination protein NusB / Antitermination factor NusB


Mass: 15838.161 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli O157:H7 (bacteria) / Gene: nusB, Z0518, ECs0469 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A782
#3: Protein/peptide 30S ribosomal protein S10 /


Mass: 12012.884 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli O157:H7 (bacteria) / Gene: rpsJ, nusE, Z4692, ECs4186 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A7R7
#5: Protein/peptide Antitermination protein N / / Regulatory protein N / PN


Mass: 12580.600 Da / Num. of mol.: 1
Source: (gene. exp.) Escherichia phage lambda (bacteriophage)
Gene: N, lambdap49 / Production host: Escherichia coli (E. coli) / References: UniProt: P03045

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

#6: Protein/peptide DNA-directed RNA polymerase subunit alpha / Polymerase / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 36558.680 Da / Num. of mol.: 2 / Source: (gene. exp.) Escherichia coli O157:H7 (bacteria) / Gene: rpoA, Z4665, ECs4160 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A7Z6, DNA-directed RNA polymerase
#7: Protein/peptide DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 10249.547 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli O157:H7 (bacteria) / Gene: rpoZ, Z5075, ECs4524 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A802, DNA-directed RNA polymerase
#8: Protein/peptide DNA-directed RNA polymerase subunit beta / Polymerase / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 150820.875 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli O157:H7 (bacteria) / Gene: rpoB, Z5560, ECs4910 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A8V4, DNA-directed RNA polymerase
#9: Protein/peptide DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 156716.141 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli O157:H7 (bacteria) / Gene: rpoC, Z5561, ECs4911 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A8T8, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules KL

#10: DNA chain DNA (I)


Mass: 20022.869 Da / Num. of mol.: 1 / Details: designed sequence / Source: (synth.) synthetic construct (others)
#11: DNA chain DNA (II)


Mass: 19919.775 Da / Num. of mol.: 1 / Details: designed sequence / Source: (synth.) synthetic construct (others)

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RNA chain , 1 types, 1 molecules R

#12: RNA chain RNA (TRANSCRIPTION BUBBLE)


Mass: 21238.717 Da / Num. of mol.: 1 / Details: designed sequence / Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)

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Non-polymers , 2 types, 3 molecules

#13: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Formula: Mg / Magnesium
#14: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Formula: Zn / Zinc

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Experimental details

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Experiment

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

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Sample preparation

Component
IDNameTypeEntity IDParent IDSource
1Structure of THE RNA POLYMERASE LAMBDA-BASED ANTITERMINATIONCOMPLEXCOMPLEX1,2,3,4,5,6,7,8,9,10,11,120MULTIPLE SOURCES
2Structure of THE RNA POLYMERASE LAMBDA-BASED ANTITERMINATIONCOMPLEXCOMPLEX1,2,3,4,5,6,7,8,9,121RECOMBINANT
3Structure of THE RNA POLYMERASE LAMBDA-BASED ANTITERMINATIONCOMPLEXCOMPLEX10,111RECOMBINANT
4Structure of THE RNA POLYMERASE LAMBDA-BASED ANTITERMINATIONCOMPLEXCOMPLEX51RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
218334Escherichia coli O157:H7 (bacteria)
3410710Escherichia phage lambda (bacteriophage)
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganism
22562Escherichia coli (E. coli)
3332630synthetic construct (others)
44562Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer ID
110 mMTris/HCl1
250 mMNaCL1
31 mMDTT1
SpecimenEmbedding applied: YES / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R3/3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 4 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 / Cs: 2.7 mm / C2 aperture diameter: 50 microns
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 69 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1EMAN2particle selection
2SerialEMimage acquisition
12RELION1.3classification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 802858
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 708030 / Algorithm: FOURIER SPACE / Symmetry type: POINT

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