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- PDB-6n60: Escherichia coli RNA polymerase sigma70-holoenzyme bound to upstr... -

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Basic information

Entry
Database: PDB / ID: 6n60
TitleEscherichia coli RNA polymerase sigma70-holoenzyme bound to upstream fork promoter DNA and Microcin J25 (MccJ25)
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • Microcin J25
  • RNA polymerase sigma factor RpoD
  • non-template strand DNA
  • template strand DNA
KeywordsTRANSFERASE/DNA / Complex / Inhibitor / Lasso peptide / Microcin J25 / RNA polymerase / TRANSFERASE-DNA complex
Function / homology
Function and homology information


RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / bacterial-type RNA polymerase core enzyme binding / regulation of DNA-templated transcription initiation / sigma factor activity / bacterial-type flagellum assembly / bacterial-type flagellum-dependent cell motility / nitrate assimilation ...RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / cytosolic DNA-directed RNA polymerase complex / bacterial-type RNA polymerase core enzyme binding / regulation of DNA-templated transcription initiation / sigma factor activity / bacterial-type flagellum assembly / bacterial-type flagellum-dependent cell motility / nitrate assimilation / transcription elongation factor complex / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription initiation / cell motility / ribonucleoside binding / : / : / : / : / : / : / DNA-directed RNA polymerase / response to heat / protein-containing complex assembly / killing of cells of another organism / intracellular iron ion homeostasis / protein dimerization activity / defense response to bacterium / response to antibiotic / magnesium ion binding / DNA binding / zinc ion binding / extracellular region / membrane / metal ion binding / cytosol / cytoplasm
Similarity search - Function
RNA polymerase sigma factor 70, non-essential domain / Sigma-70, non-essential region / RNA polymerase sigma factor 70, region 1.1 / Sigma-70 factor, region 1.1 superfamily / Sigma-70 factor, region 1.1 / RNA polymerase II, Rpb2 subunit, wall domain / RNA polymerase subunit, RPB6/omega / Eukaryotic RPB6 RNA polymerase subunit / RNA polymerase Rpb1 funnel domain / RNA polymerase, RBP11-like subunit ...RNA polymerase sigma factor 70, non-essential domain / Sigma-70, non-essential region / RNA polymerase sigma factor 70, region 1.1 / Sigma-70 factor, region 1.1 superfamily / Sigma-70 factor, region 1.1 / RNA polymerase II, Rpb2 subunit, wall domain / RNA polymerase subunit, RPB6/omega / Eukaryotic RPB6 RNA polymerase subunit / RNA polymerase Rpb1 funnel domain / RNA polymerase, RBP11-like subunit / RNA polymerase II/Efflux pump adaptor protein, barrel-sandwich hybrid domain / Sigma-70 factors family signature 1. / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / Topoisomerase I; Chain A, domain 4 / DNA-directed RNA polymerase, insert domain / : / RNA polymerase sigma-70 region 1.2 / Sigma-70 factor, region 1.2 / RNA polymerase sigma-70 region 3 / Sigma-70 region 3 / Sigma-70 factors family signature 2. / RNA polymerase sigma-70 / RNA Polymerase Alpha Subunit; Chain A, domain 2 / RNA polymerase sigma-70 region 4 / Sigma-70, region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 like domain / Sigma-70 region 2 / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like / Gyrase A; domain 2 / 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 / Beta Complex / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / 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 Rpb1, clamp domain superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / 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 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / 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 / OB fold (Dihydrolipoamide Acetyltransferase, E2P) / Winged helix-like DNA-binding domain superfamily / Alpha-Beta Complex / Beta Barrel / 2-Layer Sandwich / Orthogonal Bundle / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
Microcin J25 / DNA / DNA (> 10) / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / RNA polymerase sigma factor RpoD / Microcin J25
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.68 Å
AuthorsBraffman, N. / Hauver, J. / Campbell, E.A. / Darst, S.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM118130 United States
CitationJournal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2019
Title: Structural mechanism of transcription inhibition by lasso peptides microcin J25 and capistruin.
Authors: Braffman, N.R. / Piscotta, F.J. / Hauver, J. / Campbell, E.A. / Link, A.J. / Darst, S.A.
History
DepositionNov 23, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 9, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 23, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.pdbx_database_id_PubMed ..._citation.journal_abbrev / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Feb 6, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Feb 27, 2019Group: Data collection / Structure summary / Category: pdbx_molecule_features
Revision 1.4Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Oct 11, 2023Group: 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

Downloads & links

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Assembly

Deposited unit
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: DNA-directed RNA polymerase subunit omega
F: RNA polymerase sigma factor RpoD
M: Microcin J25
N: non-template strand DNA
T: template strand DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)458,37412
Polymers458,2199
Non-polymers1553
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area43950 Å2
ΔGint-232 kcal/mol
Surface area156730 Å2
MethodPISA
Unit cell
Length a, b, c (Å)172.906, 172.906, 387.263
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number92
Space group name H-MP41212

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Components

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

#1: Protein DNA-directed RNA polymerase subunit alpha / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 26459.125 Da / Num. of mol.: 2 / Fragment: N-terminal domain (UNP residues 1-234)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoA, pez, phs, sez, b3295, JW3257 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A7Z4, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 150820.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: rpoB, groN, nitB, rif, ron, stl, stv, tabD, b3987, JW3950
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A8V2, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 155576.984 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoC, tabB, b3988, JW3951 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A8T7, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 10249.547 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoZ, b3649, JW3624 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0A800, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules NT

#7: DNA chain non-template strand DNA


Mass: 8924.757 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#8: DNA chain template strand DNA


Mass: 7363.770 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Protein / Protein/peptide , 2 types, 2 molecules FM

#5: Protein RNA polymerase sigma factor RpoD / Sigma-70


Mass: 70238.102 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoD / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q0P6L9
#6: Protein/peptide Microcin J25 / MccJ25


Type: Polypeptide / Class: Inhibitor / Mass: 2126.348 Da / Num. of mol.: 1 / Fragment: UNP residues 38-58
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: mcjA / Production host: Escherichia coli (E. coli) / References: UniProt: Q9X2V7, Microcin J25

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Non-polymers , 2 types, 3 molecules

#9: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#10: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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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: 3.26 Å3/Da / Density % sol: 62.23 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 6.8
Details: 0.1 M HEPES, pH 6.7, 0.2 M magnesium chloride, 7% w/v PEG3350, 4% v/v glycerol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.9763 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Jul 10, 2015
RadiationMonochromator: Cryo-cooled double crystal Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 3.68→50 Å / Num. obs: 64026 / % possible obs: 98 % / Redundancy: 9.2 % / Rpim(I) all: 0.06 / Net I/σ(I): 10.14
Reflection shellResolution: 3.68→3.81 Å / Redundancy: 4.3 % / Mean I/σ(I) obs: 0.55 / Num. unique obs: 5759 / CC1/2: 0.144 / Rpim(I) all: 1.39 / % possible all: 91

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Processing

Software
NameVersionClassification
PHENIX(1.13_2998: ???)refinement
HKL-2000data reduction
HKL-2000data scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 4LJZ

4ljz


Resolution: 3.68→49.551 Å / SU ML: 0.71 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 36.15
RfactorNum. reflection% reflection
Rfree0.3062 1945 3.07 %
Rwork0.2634 --
obs0.2647 63407 98.35 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 3.68→49.551 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms26615 1087 3 0 27705
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00328218
X-RAY DIFFRACTIONf_angle_d0.68438357
X-RAY DIFFRACTIONf_dihedral_angle_d16.87817160
X-RAY DIFFRACTIONf_chiral_restr0.0434403
X-RAY DIFFRACTIONf_plane_restr0.0044841
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.68-3.7720.36761200.36343926X-RAY DIFFRACTION90
3.772-3.8740.40651360.3574210X-RAY DIFFRACTION96
3.874-3.98790.38611330.34424317X-RAY DIFFRACTION98
3.9879-4.11660.35561430.32464399X-RAY DIFFRACTION100
4.1166-4.26360.37511380.3014359X-RAY DIFFRACTION100
4.2636-4.43420.36671360.29734426X-RAY DIFFRACTION100
4.4342-4.63590.28161440.27944395X-RAY DIFFRACTION100
4.6359-4.88010.32551400.26454421X-RAY DIFFRACTION100
4.8801-5.18560.34951380.26774418X-RAY DIFFRACTION100
5.1856-5.58540.33261430.27474472X-RAY DIFFRACTION100
5.5854-6.14660.3461420.2734448X-RAY DIFFRACTION99
6.1466-7.03390.32791410.2754452X-RAY DIFFRACTION99
7.0339-8.85370.28821450.23924523X-RAY DIFFRACTION99
8.8537-49.55510.25141460.2334696X-RAY DIFFRACTION98

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