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- PDB-5lm7: Crystal structure of the lambda N-Nus factor complex -

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Basic information

Entry
Database: PDB / ID: 5lm7
TitleCrystal structure of the lambda N-Nus factor complex
Components
  • 30S ribosomal protein S10
  • Antitermination protein N
  • N utilization substance protein B homolog
  • RNA (29-MER)
  • Transcription termination/antitermination protein NusA
KeywordsTRANSCRIPTION / Transcription regulation antitermination Nus proteins Phage lambda N protein
Function / homology
Function and homology information


bacterial-type RNA polymerase core enzyme binding / RNA polymerase binding / transcription antitermination factor activity, RNA binding / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription termination / RNA stem-loop binding / small ribosomal subunit / tRNA binding / single-stranded RNA binding ...bacterial-type RNA polymerase core enzyme binding / RNA polymerase binding / transcription antitermination factor activity, RNA binding / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription termination / RNA stem-loop binding / small ribosomal subunit / tRNA binding / single-stranded RNA binding / structural constituent of ribosome / translation / DNA-binding transcription factor activity / nucleotide binding / regulation of DNA-templated transcription / regulation of transcription by RNA polymerase II / DNA binding / RNA binding / cytosol
Similarity search - Function
Antitermination protein N, arginine-rich motif / 36-mer N-terminal peptide of the N protein (N36) / N-utilizing Substance Protein B Homolog; Chain A / NusB-like / NusB antitermination factor / NusB/RsmB/TIM44 / NusB family / NusB-like superfamily / Transcription termination factor NusA, C-terminal duplication / Transcription termination factor NusA ...Antitermination protein N, arginine-rich motif / 36-mer N-terminal peptide of the N protein (N36) / N-utilizing Substance Protein B Homolog; Chain A / NusB-like / 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 / Ribosomal protein S10 / 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 / 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 / Alpha-Beta Plaits / Nucleic acid-binding, OB-fold / 2-Layer Sandwich / Orthogonal Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
RNA / RNA (> 10) / Small ribosomal subunit protein uS10 / Transcription antitermination protein NusB / Antitermination protein N / Transcription termination/antitermination protein NusA
Similarity search - Component
Biological speciesEscherichia coli O157:H7 (bacteria)
Escherichia coli O45:K1 (bacteria)
Enterobacteria phage lambda (virus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.35 Å
AuthorsSaid, N. / Santos, K. / Weber, G. / 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
B: N utilization substance protein B homolog
J: 30S ribosomal protein S10
N: Antitermination protein N
R: RNA (29-MER)
C: Transcription termination/antitermination protein NusA
D: N utilization substance protein B homolog
E: 30S ribosomal protein S10
F: Antitermination protein N
G: RNA (29-MER)


Theoretical massNumber of molelcules
Total (without water)191,05110
Polymers191,05110
Non-polymers00
Water00
1
A: Transcription termination/antitermination protein NusA
B: N utilization substance protein B homolog
J: 30S ribosomal protein S10
N: Antitermination protein N
R: RNA (29-MER)


Theoretical massNumber of molelcules
Total (without water)95,5255
Polymers95,5255
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12790 Å2
ΔGint-35 kcal/mol
Surface area48230 Å2
MethodPISA
2
C: Transcription termination/antitermination protein NusA
D: N utilization substance protein B homolog
E: 30S ribosomal protein S10
F: Antitermination protein N
G: RNA (29-MER)


Theoretical massNumber of molelcules
Total (without water)95,5255
Polymers95,5255
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12320 Å2
ΔGint-32 kcal/mol
Surface area45910 Å2
MethodPISA
Unit cell
Length a, b, c (Å)95.776, 101.803, 279.918
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Transcription termination/antitermination protein NusA


Mass: 47666.641 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli O157:H7 (bacteria) / Gene: nusA, Z4530, ECs4050 / Production host: Escherichia coli (E. coli) / References: UniProt: P0AFF8
#2: Protein N utilization substance protein B homolog / Protein NusB


Mass: 15838.161 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli O45:K1 (strain S88 / ExPEC) (bacteria)
Strain: S88 / ExPEC / Gene: nusB, ECS88_0411 / Production host: Escherichia coli (E. coli) / References: UniProt: B7MD74
#3: Protein 30S ribosomal protein S10


Mass: 12167.051 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli O45:K1 (strain S88 / ExPEC) (bacteria)
Strain: S88 / ExPEC / Gene: rpsJ, ECS88_3708 / Production host: Escherichia coli (E. coli) / References: UniProt: B7MCT6
#4: Protein Antitermination protein N / Regulatory protein N / PN


Mass: 10279.788 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterobacteria phage lambda (virus) / Gene: N, lambdap49 / Production host: Escherichia coli (E. coli) / References: UniProt: P03045
#5: RNA chain RNA (29-MER)


Mass: 9573.745 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Enterobacteria phage lambda (virus)

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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: 3.57 Å3/Da / Density % sol: 65.56 %
Crystal growTemperature: 295 K / Method: vapor diffusion, sitting drop
Details: 0.1M HEPES pH 7.5, 40%(v/v) ethylene glycol, 5% (w/v) PEG 3000

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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: Jan 29, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.918 Å / Relative weight: 1
ReflectionResolution: 3.35→50 Å / Num. obs: 40077 / % possible obs: 99.3 % / Redundancy: 7.3 % / Rsym value: 0.15 / Net I/σ(I): 11.56
Reflection shellResolution: 3.35→3.55 Å / Redundancy: 7.5 % / Rsym value: 2.02 / % possible all: 98.3

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Processing

Software
NameVersionClassification
PHENIX(1.10.1_2155: ???)refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1L2F, 2KWP, 1U9L, 3B3D, 1QFQ

1l2f

2kwp

1u9l

3b3d

1qfq


Resolution: 3.35→39.52 Å / SU ML: 0.62 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 42.66 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.3354 2000 5 %
Rwork0.2966 --
obs0.2986 39986 99.38 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 3.35→39.52 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms11642 1193 0 0 12835
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00413123
X-RAY DIFFRACTIONf_angle_d0.84518012
X-RAY DIFFRACTIONf_dihedral_angle_d23.3698018
X-RAY DIFFRACTIONf_chiral_restr0.0482126
X-RAY DIFFRACTIONf_plane_restr0.0052159
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.35-3.43370.48491400.45842665X-RAY DIFFRACTION99
3.4337-3.52650.46361400.43482651X-RAY DIFFRACTION99
3.5265-3.63020.40711400.3922668X-RAY DIFFRACTION99
3.6302-3.74730.44041400.38332663X-RAY DIFFRACTION99
3.7473-3.88110.44361410.35532680X-RAY DIFFRACTION99
3.8811-4.03640.40321410.35742667X-RAY DIFFRACTION99
4.0364-4.21990.39111420.32292707X-RAY DIFFRACTION100
4.2199-4.44210.33611420.30032696X-RAY DIFFRACTION100
4.4421-4.720.30831430.28372705X-RAY DIFFRACTION100
4.72-5.08370.31241440.29672736X-RAY DIFFRACTION100
5.0837-5.5940.37591440.31092740X-RAY DIFFRACTION100
5.594-6.40050.3791450.31582749X-RAY DIFFRACTION100
6.4005-8.05280.29781460.27512782X-RAY DIFFRACTION100
8.0528-39.52310.24861520.21962877X-RAY DIFFRACTION99

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