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- PDB-5lm9: Structure of E. coli NusA -

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Basic information

Entry
Database: PDB / ID: 5lm9
TitleStructure of E. coli NusA
ComponentsTranscription termination/antitermination protein NusA
KeywordsTRANSCRIPTION / Transcription factor / Antitermination Termination / Nus proteins
Function / homology
Function and homology information


bacterial-type RNA polymerase core enzyme binding / transcription elongation factor complex / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription termination / ribosome biogenesis / protein complex oligomerization / DNA-binding transcription factor activity / protein domain specific binding / nucleotide binding ...bacterial-type RNA polymerase core enzyme binding / transcription elongation factor complex / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription termination / ribosome biogenesis / protein complex oligomerization / DNA-binding transcription factor activity / protein domain specific binding / nucleotide binding / RNA binding / cytosol
Similarity search - Function
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 / Type-1 KH domain profile. / DNA repair Rad51/transcription factor NusA, alpha-helical ...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 / Type-1 KH domain profile. / DNA repair Rad51/transcription factor NusA, alpha-helical / Helix-hairpin-helix domain / S1 domain profile. / Ribosomal protein S1-like RNA-binding domain / S1 RNA binding domain / S1 domain / K homology domain superfamily, prokaryotic type / K homology domain-like, alpha/beta / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
Transcription termination/antitermination protein NusA
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.143 Å
AuthorsSaid, N. / Weber, G. / Santos, K. / Wahl, M.C.
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
DepositionJul 29, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 5, 2017Provider: repository / Type: Initial release
Revision 1.1Dec 6, 2017Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jan 10, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Transcription termination/antitermination protein NusA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)36,8007
Polymers36,2951
Non-polymers5056
Water1,74797
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area820 Å2
ΔGint-55 kcal/mol
Surface area19590 Å2
MethodPISA
Unit cell
Length a, b, c (Å)74.688, 147.182, 52.387
Angle α, β, γ (deg.)90.00, 112.56, 90.00
Int Tables number5
Space group name H-MC121
Components on special symmetry positions
IDModelComponents
11A-660-

HOH

21A-694-

HOH

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Components

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


Mass: 36295.043 Da / Num. of mol.: 1 / Fragment: UNP residues 101-426
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
#2: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: SO4
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 97 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.67 Å3/Da / Density % sol: 66.52 %
Crystal growTemperature: 295 K / Method: vapor diffusion, sitting drop / Details: 0.1 M HEPES, pH 7.5, 0.1 M NaCl, 1.6 M (NH4)2SO4

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: BESSY / Beamline: 14.1 / Wavelength: 0.918 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Oct 5, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.918 Å / Relative weight: 1
ReflectionResolution: 2.14→50 Å / Num. obs: 28443 / % possible obs: 99.1 % / Redundancy: 6.8 % / Rsym value: 0.01 / Net I/σ(I): 12.15
Reflection shellResolution: 2.14→2.27 Å

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Processing

Software
NameVersionClassification
PHENIX1.9_1692refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1L2F

1l2f


Resolution: 2.143→46.949 Å / SU ML: 0.37 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 31.34
RfactorNum. reflection% reflection
Rfree0.244 1423 5.01 %
Rwork0.2145 --
obs0.2161 28428 99.35 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.143→46.949 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2474 0 26 97 2597
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0032541
X-RAY DIFFRACTIONf_angle_d0.6383445
X-RAY DIFFRACTIONf_dihedral_angle_d13.284962
X-RAY DIFFRACTIONf_chiral_restr0.026398
X-RAY DIFFRACTIONf_plane_restr0.003457
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.1431-2.21970.38261350.36612563X-RAY DIFFRACTION95
2.2197-2.30860.40071420.33842698X-RAY DIFFRACTION100
2.3086-2.41370.34771440.29512727X-RAY DIFFRACTION100
2.4137-2.54090.31251420.28192685X-RAY DIFFRACTION100
2.5409-2.70010.26041420.25732713X-RAY DIFFRACTION100
2.7001-2.90850.32571440.24852731X-RAY DIFFRACTION100
2.9085-3.20120.31161430.24612710X-RAY DIFFRACTION100
3.2012-3.66420.24371420.21412705X-RAY DIFFRACTION100
3.6642-4.61590.20361440.17612724X-RAY DIFFRACTION100
4.6159-46.96050.18141450.17132749X-RAY DIFFRACTION100

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