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- EMDB-0043: cryoEM structure of the lambdaN Transcription Antitermination Com... -

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Basic information

Entry
Database: EMDB / ID: EMD-0043
TitlecryoEM structure of the lambdaN Transcription Antitermination Complex at 3.7 A resolution.
Map datacryoEM of the lambdaN Transcription Antitermination Complex, 3.7A map
Sample
  • Complex: Transcription Antitermination Complex
Function / homology
Function and homology information


RNA polymerase binding / transcription elongation-coupled chromatin remodeling / bacterial-type RNA polymerase core enzyme binding / transcription antitermination factor activity, RNA binding / DNA-directed RNA polymerase complex / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription termination / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity ...RNA polymerase binding / transcription elongation-coupled chromatin remodeling / bacterial-type RNA polymerase core enzyme binding / transcription antitermination factor activity, RNA binding / DNA-directed RNA polymerase complex / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription termination / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / RNA stem-loop binding / tRNA binding / single-stranded RNA binding / protein dimerization activity / ribosome / structural constituent of ribosome / ribonucleoprotein complex / translation / DNA-binding transcription factor activity / nucleotide binding / DNA-templated transcription / regulation of transcription by RNA polymerase II / magnesium ion binding / DNA binding / RNA binding / zinc ion binding / 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 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. / DNA repair Rad51/transcription factor NusA, alpha-helical / Type-1 KH domain profile. / 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, alpha subunit / DNA-directed RNA polymerase beta subunit, bacterial-type / Ribosomal protein S1-like RNA-binding domain / S1 RNA binding domain / S1 domain / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / 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, 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 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / 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
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 coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsKrupp F / Spahn 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 cryo- ...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.
History
DepositionMay 30, 2018-
Header (metadata) releaseAug 22, 2018-
Map releaseFeb 20, 2019-
UpdateApr 17, 2019-
Current statusApr 17, 2019Processing site: PDBe / Status: Released

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Structure visualization

Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0043.png

Downloads & links

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Map

FileDownload / File: emd_0043.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationcryoEM of the lambdaN Transcription Antitermination Complex, 3.7A map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 240 pix.
= 324. Å		1.35 Å/pix.
x 240 pix.
= 324. Å		1.35 Å/pix.
x 240 pix.
= 324. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0138
Minimum - Maximum-0.08612833 - 0.15245315
Average (Standard dev.)0.00035626657 (±0.0039406037)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-120-120-120
Dimensions240240240
Spacing240240240
CellA=B=C: 324.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_0043_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: cryoEM of the lambdaN Transcription Antitermination Complex, 1st...

Fileemd_0043_half_map_1.map
AnnotationcryoEM of the lambdaN Transcription Antitermination Complex, 1st half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: cryoEM of the lambdaN Transcription Antitermination Complex, 2nd...

Fileemd_0043_half_map_2.map
AnnotationcryoEM of the lambdaN Transcription Antitermination Complex, 2nd half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Transcription Antitermination Complex

EntireName: Transcription Antitermination Complex
Components
  • Complex: Transcription Antitermination Complex

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Supramolecule #1: Transcription Antitermination Complex

SupramoleculeName: Transcription Antitermination Complex / type: complex / ID: 1 / Parent: 0
Details: Consisting of bacterial RNA Polymerase, NusA, NusB, NusE, NusG, DNA, RNA and lambdaN
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 500 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
Component:
ConcentrationFormulaName
10.0 mMHEPES-NaOHHEPES-NaOH
50.0 mMNaClsodium chloride
1.0 mMDTTDTT
GridModel: Quantifoil R2/4 / Material: COPPER/RHODIUM / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER
Details: A thin carbon support film was placed over the holey carbon film.
VitrificationCryogen name: ETHANE / Chamber humidity: 75 % / Chamber temperature: 4 K / Instrument: FEI VITROBOT MARK II

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Average exposure time: 20.0 sec. / Average electron dose: 69.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 805023
CTF correctionSoftware - Name: SPHIRE / Software - details: CTER
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPHIRE / Software - details: sxmeridien / Number images used: 708030
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE / Software - details: sxmeridien
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: SPHIRE / Software - details: sxmeridien
Final 3D classificationSoftware - Name: SPARX / Software - details: own script

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 97.43 / Target criteria: Cross-correlation coefficient
Output model

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

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