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- PDB-5ms0: pseudo-atomic model of the RNA polymerase lambda-based antitermin... -

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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 ...Polymerase) x 4
  • (Transcription termination/antitermination protein ...) x 2
  • 30S ribosomal protein S10
  • Antitermination protein
  • DNAI
  • DNAII
  • N utilization substance protein B homolog
  • RNA transcription bubble
  • nascent RNA
KeywordsTRANSCRIPTION/DNA/RNA / DNA-DEPENDENT RNA POLYMERASE / BACTERIAL TRANSCRIPTION / TERNARY ELONGATION COMPLEX / ANTITERMINATION / TRANSCRIPTION-DNA-RNA complex
Function/homologyAntitermination protein N, arginine-rich motif / 36-mer N-terminal peptide of the N protein (N36) / transcription termination from bacterial-type RNA polymerase promoter / Transcription termination factor NusA, C-terminal duplication / Transcription termination factor nusG signature. / Transcription antitermination protein, NusG, bacteria, conserved site / Transcription antitermination protein, NusG / Transcription factor NusA, N-terminal / NusA, N-terminal domain superfamily / Transcription termination factor NusA ...Antitermination protein N, arginine-rich motif / 36-mer N-terminal peptide of the N protein (N36) / transcription termination from bacterial-type RNA polymerase promoter / Transcription termination factor NusA, C-terminal duplication / Transcription termination factor nusG signature. / Transcription antitermination protein, NusG, bacteria, conserved site / Transcription antitermination protein, NusG / Transcription factor NusA, N-terminal / NusA, N-terminal domain superfamily / Transcription termination factor NusA / NusB antitermination factor / KH domain, NusA-like / Transcription termination/antitermination protein NusA, bacterial / NusB/RsmB/TIM44 / NusB-like superfamily / NusA N-terminal domain / NusA-like KH domain / Transcription termination factor nusG / bacterial-type RNA polymerase core enzyme binding / NusG, N-terminal / NusB family / NusG, N-terminal domain superfamily / regulation of DNA-templated transcription, elongation / RNA polymerase complex / RNA stem-loop binding / DNA-directed RNA polymerase complex / DNA-templated transcription, elongation / Type-1 KH domain profile. / Helix-hairpin-helix domain / S1 domain profile. / DNA-directed RNA polymerase, omega subunit / DNA repair Rad51/transcription factor NusA, alpha-helical / DNA-directed RNA polymerase, subunit beta-prime / DNA-directed RNA polymerase beta subunit, bacterial-type / DNA-directed RNA polymerase, beta subunit, external 1 domain / DNA-directed RNA polymerase, alpha subunit / RNA polymerase, alpha subunit, C-terminal / transcription antitermination factor activity, RNA binding / RNA-binding domain, S1 / S1 domain / Bacterial RNA polymerase, alpha chain C terminal domain / RNA polymerase beta subunit external 1 domain / transcription antitermination / RNA polymerase subunit, RPB6/omega / RPB6/omega subunit-like superfamily / RNA polymerase, subunit omega/K/RPB6 / RNA polymerase, beta subunit, protrusion / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerases beta chain signature. / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 7 / RNA polymerase, beta subunit, conserved site / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / DNA-directed RNA polymerase, subunit 2 / RNA polymerase Rpb2, OB-fold / S1 RNA binding domain / ribonucleoside binding / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 3 / RNA polymerase, N-terminal / DNA-templated transcription, termination / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / RNA polymerase Rpb6 / RNA polymerase beta subunit / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, domain 6 / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / Ribosomal protein S10, conserved site / Ribosomal protein S10 signature. / Ribosomal protein S10 / Ribosomal protein S10 domain superfamily / Ribosomal protein S10 domain / K homology domain superfamily, prokaryotic type / K homology domain-like, alpha/beta / cytosolic small ribosomal subunit / Translation protein SH3-like domain superfamily / KOW / Ribosomal protein L2, domain 2 / go:0006461: / Ribosomal protein S10p/S20e / structural constituent of ribosome / KOW motif
Function and homology information
Specimen sourceEscherichia coli / / bacteria /
MethodElectron microscopy (9.8 Å resolution / Particle / Single particle) / Transmission electron microscopy
AuthorsSaid, N. / Krupp, F.
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 / 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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Dec 29, 2016 / Release: May 3, 2017
RevisionDateData content typeGroupProviderType
1.0May 3, 2017Structure modelrepositoryInitial release
1.1May 10, 2017Structure modelDatabase references

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Structure visualization

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Assembly

Deposited unit
N: Antitermination protein
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: N utilization substance protein B homolog
M: Transcription termination/antitermination protein NusA
O: DNA-directed RNA polymerase subunit omega
hetero molecules


Theoretical massNumber of molelcules
Total (without water)537,67017
Polyers537,51514
Non-polymers1553
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein/peptide , 3 types, 3 molecules NEL

#1: Protein/peptide Antitermination protein


Mass: 9877.343 Da / Num. of mol.: 1 / Details: lambda N factor / Source: (gene. exp.) Escherichia coli / Gene: AKG99_28260 / Production host: Escherichia coli / References: UniProt:A0A0L6XR33, UniProt:P03045*PLUS
#6: Protein/peptide 30S ribosomal protein S10 / / Small ribosomal subunit protein uS10


Mass: 11340.067 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli / Gene: rpsJ, nusE, b3321, JW3283 / Production host: Escherichia coli / References: UniProt:P0A7R5
#11: Protein/peptide N utilization substance protein B homolog / Protein NusB


Mass: 15709.967 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli / Gene: nusB, ECS88_0411 / Production host: Escherichia coli / References: UniProt:B7MD74, UniProt:P0A780*PLUS

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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: (gene. exp.) Escherichia coli / Production host: Escherichia coli
#8: RNA chain RNA transcription bubble


Mass: 4492.788 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli / Production host: Escherichia coli

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

#3: 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 / Gene: rpoA, pez, phs, sez, b3295, JW3257 / Production host: Escherichia coli
References: UniProt:P0A7Z4, EC:2.7.7.6 (DNA-directed RNA polymerase)
#4: Protein/peptide DNA-directed RNA polymerase subunit beta / Polymerase / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 150804.922 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli
Gene: rpoB, groN, nitB, rif, ron, stl, stv, tabD, b3987, JW3950
Production host: Escherichia coli
References: UniProt:P0A8V2, EC:2.7.7.6 (DNA-directed RNA polymerase)
#5: Protein/peptide DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 156537.031 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli / Gene: rpoC, Z5561, ECs4911 / Production host: Escherichia coli
References: UniProt:P0A8T8, UniProt:P0A8T7*PLUS, EC:2.7.7.6 (DNA-directed RNA polymerase)
#13: 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 / Gene: rpoZ, b3649, JW3624 / Production host: Escherichia coli
References: UniProt:P0A800, EC:2.7.7.6 (DNA-directed RNA polymerase)

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

#7: Protein/peptide Transcription termination/antitermination protein NusG


Mass: 20688.654 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli / Gene: nusG, b3982, JW3945 / Production host: Escherichia coli / References: UniProt:P0AFG0
#12: Protein/peptide Transcription termination/antitermination protein NusA / N utilization substance protein A / Transcription termination/antitermination L factor


Mass: 55059.828 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli / Gene: nusA, b3169, JW3138 / Production host: Escherichia 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: (gene. exp.) Escherichia coli / Production host: Escherichia coli
#10: DNA chain DNAII


Mass: 11942.696 Da / Num. of mol.: 1 / Source: (gene. exp.) Escherichia coli / Production host: Escherichia 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 / Formula: Mg / : Magnesium
#15: 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

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Sample preparation

ComponentName: lambdaN-dependent antitermination complex / Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli
Source (recombinant)Organism: Escherichia coli
Buffer solutionpH: 7.5
SpecimenDetails: 10 mM HEPES-NaOH, pH 7.5, 50 mM NaCl, 1 mM DTT / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyMicroscope model: 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
3D reconstructionResolution: 9.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 23983 / Symmetry type: POINT

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