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- EMDB-3561: map of the RNA polymerase lambda-based antitermination complex so... -

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Basic information

Entry
Database: EMDB / ID: 3561
Titlemap of the RNA polymerase lambda-based antitermination complex solved by cryo-EM
Map data
SamplelambdaN-dependent antitermination complex
  • Antitermination protein
  • (nucleic-acidNucleic acid) x 4
  • (DNA-directed RNA polymerase subunit ...) x 4
  • 30S ribosomal protein S10
  • (Transcription termination/antitermination protein ...) x 2
  • N utilization substance protein B homolog
  • (ligand) x 2
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 antitermination protein, NusG / Transcription antitermination protein, NusG, bacteria, conserved site / Transcription termination factor nusG signature. / NusB antitermination factor / Transcription factor NusA, N-terminal / NusA, N-terminal domain superfamily ...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 antitermination protein, NusG / Transcription antitermination protein, NusG, bacteria, conserved site / Transcription termination factor nusG signature. / NusB antitermination factor / Transcription factor NusA, N-terminal / NusA, N-terminal domain superfamily / Transcription termination/antitermination protein NusA, bacterial / KH domain, NusA-like / Transcription termination factor NusA / 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. / 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, external 1 domain / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase, alpha subunit, C-terminal / DNA-directed RNA polymerase, alpha subunit / transcription antitermination factor activity, RNA binding / S1 domain / RNA-binding domain, S1 / Bacterial RNA polymerase, alpha chain C terminal domain / RNA polymerase beta subunit external 1 domain / transcription antitermination / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase subunit, RPB6/omega / RPB6/omega subunit-like superfamily / RNA polymerase, subunit omega/K/RPB6 / RNA polymerase Rpb2, domain 3 / RNA polymerase, beta subunit, protrusion / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 7 / RNA polymerases beta chain signature. / RNA polymerase Rpb2, OB-fold / DNA-directed RNA polymerase, subunit 2 / RNA polymerase, beta subunit, conserved site / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / ribonucleoside binding / S1 RNA binding domain / 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 Rpb1, domain 4 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 5 / RNA polymerase, N-terminal / DNA-templated transcription, termination / DNA-directed RNA polymerase / DNA-directed 5'-3' RNA polymerase activity / 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 signature. / Ribosomal protein S10, conserved site / Ribosomal protein S10 / Ribosomal protein S10 domain / Ribosomal protein S10 domain superfamily / K homology domain superfamily, prokaryotic type / K homology domain-like, alpha/beta / Translation protein SH3-like domain superfamily / KOW / cytosolic small ribosomal subunit / Ribosomal protein L2, domain 2 / Ribosomal protein S10p/S20e / KOW motif / structural constituent of ribosome / Nucleic acid-binding, OB-fold
Function and homology information
SourceEscherichia coli / / bacteria /
Methodsingle particle reconstruction / cryo EM / 9.8 Å resolution
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 ReportPDB-ID: 5ms0

SummaryFull reportAbout validation report
DateDeposition: Dec 29, 2016 / Header (metadata) release: May 3, 2017 / Map release: May 3, 2017 / Last update: May 10, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.007
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.007
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-5ms0
  • Surface level: 0.007
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_3561.map.gz (map file in CCP4 format, 55297 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
240 pix
1.28 Å/pix.
= 307.2 Å
240 pix
1.28 Å/pix.
= 307.2 Å
240 pix
1.28 Å/pix.
= 307.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.28 Å
Density
Contour Level:0.007 (by emdb), 0.007 (movie #1):
Minimum - Maximum-0.012556402 - 0.028230608
Average (Standard dev.)0.0003642441 (0.0021255864)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions240240240
Origin-120-120-120
Limit119119119
Spacing240240240
CellA=B=C: 1 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.281.281.28
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z307.200307.200307.200
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS-120-120-120
NC/NR/NS240240240
D min/max/mean-0.0130.0280.000

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Supplemental data

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Sample components

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Entire lambdaN-dependent antitermination complex

EntireName: lambdaN-dependent antitermination complex / Number of components: 16

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Component #1: protein, lambdaN-dependent antitermination complex

ProteinName: lambdaN-dependent antitermination complex / Recombinant expression: No
SourceSpecies: Escherichia coli
Source (engineered)Expression System: Escherichia coli

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Component #2: protein, Antitermination protein

ProteinName: Antitermination protein / Details: lambda N factor / Recombinant expression: No
MassTheoretical: 9.877343 kDa
SourceSpecies:
Source (engineered)Expression System: Escherichia coli

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Component #3: nucleic-acid, nascent RNA

Nucleic-acidName: nascent RNA / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
AUCUUUAAAA AUAAGCCCUG AAGAAGGGC
MassTheoretical: 9.33965 kDa
SourceSpecies:

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Component #4: protein, DNA-directed RNA polymerase subunit alpha

ProteinName: DNA-directed RNA polymerase subunit alpha / Recombinant expression: No
MassTheoretical: 36.55868 kDa
SourceSpecies:
Source (engineered)Expression System: Escherichia coli

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Component #5: protein, DNA-directed RNA polymerase subunit beta

ProteinName: DNA-directed RNA polymerase subunit beta / Recombinant expression: No
MassTheoretical: 150.804922 kDa
SourceSpecies:
Source (engineered)Expression System: Escherichia coli

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Component #6: protein, DNA-directed RNA polymerase subunit beta'

ProteinName: DNA-directed RNA polymerase subunit beta' / Recombinant expression: No
MassTheoretical: 156.537031 kDa
SourceSpecies:
Source (engineered)Expression System: Escherichia coli

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Component #7: protein, 30S ribosomal protein S10

ProteinName: 30S ribosomal protein S10 / Recombinant expression: No
MassTheoretical: 11.340067 kDa
SourceSpecies:
Source (engineered)Expression System: Escherichia coli

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Component #8: protein, Transcription termination/antitermination protein NusG

ProteinName: Transcription termination/antitermination protein NusG
Recombinant expression: No
MassTheoretical: 20.688654 kDa
SourceSpecies:
Source (engineered)Expression System: Escherichia coli

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Component #9: nucleic-acid, RNA transcription bubble

Nucleic-acidName: RNA transcription bubble / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
CAUAAAGACC AGGC
MassTheoretical: 4.492788 kDa
SourceSpecies:

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Component #10: nucleic-acid, DNAI

Nucleic-acidName: DNAI / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DG)(DG)(DC)(DA)(DG)(DT)(DA)(DC)(DT)(DA) (DG)(DT)(DA)(DA)(DA)(DG)(DT)(DA)(DC)(DT) (DT)(DG)(DA)(DG)(DC)(DT)(DT)
MassTheoretical: 8.355408 kDa
SourceSpecies:

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Component #11: nucleic-acid, DNAII

Nucleic-acidName: DNAII / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DA)(DA)(DG)(DC)(DT)(DC)(DA)(DA)(DG)(DT) (DA)(DC)(DT)(DT)(DA)(DA)(DG)(DC)(DC)(DT) (DG)(DG)(DT)(DC)(DA)(DT)(DT)(DA)(DC)(DT) (DA)(DG)(DT)(DA)(DC)(DT)(DG)(DC)(DC)
MassTheoretical: 11.942696 kDa
SourceSpecies:

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Component #12: protein, N utilization substance protein B homolog

ProteinName: N utilization substance protein B homolog / Recombinant expression: No
MassTheoretical: 15.709967 kDa
SourceSpecies:
Source (engineered)Expression System: Escherichia coli

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Component #13: protein, Transcription termination/antitermination protein NusA

ProteinName: Transcription termination/antitermination protein NusA
Recombinant expression: No
MassTheoretical: 55.059828 kDa
SourceSpecies:
Source (engineered)Expression System: Escherichia coli

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Component #14: protein, DNA-directed RNA polymerase subunit omega

ProteinName: DNA-directed RNA polymerase subunit omega / Recombinant expression: No
MassTheoretical: 10.249547 kDa
SourceSpecies:
Source (engineered)Expression System: Escherichia coli

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Component #15: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 2.430505 MDa
SourceSpecies:

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Component #16: ligand, ZINC ION

LigandName: ZINC ION / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 6.540905 MDa
SourceSpecies:

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 23983
3D reconstructionResolution: 9.8 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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