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Basic information

Entry
Database: PDB / ID: 6omf
TitleCryoEM structure of SigmaS-transcription initiation complex with activator Crl
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • Non-template DNA strand
  • RNA polymerase sigma factor RpoS
  • Sigma factor-binding protein Crl
  • Template DNA strand
KeywordsTRANSCRIPTION / Transferase/DNA / Transcription-activator / DNA/RNA / SigmaS / beta' / Transferase-DNA complex
Function / homology
Function and homology information


positive regulation of cellulose biosynthetic process / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / regulation of DNA-templated transcription initiation / sigma factor activity / bacterial-type flagellum assembly / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / bacterial-type flagellum-dependent cell motility ...positive regulation of cellulose biosynthetic process / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / regulation of DNA-templated transcription initiation / sigma factor activity / bacterial-type flagellum assembly / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / bacterial-type flagellum-dependent cell motility / nitrate assimilation / nucleotidyltransferase activity / DNA-directed RNA polymerase complex / 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 / intracellular iron ion homeostasis / protein dimerization activity / response to antibiotic / positive regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
Sigma factor-binding protein Crl monomer / Sigma factor-binding transcriptional regulator Crl / Sigma factor-binding protein Crl superfamily / Sigma factor-binding transcriptional regulator Crl / RNA polymerase sigma factor RpoS / RNA polymerase II, Rpb2 subunit, wall domain / TATA-Binding Protein / RNA polymerase subunit, RPB6/omega / Eukaryotic RPB6 RNA polymerase subunit / RNA polymerase Rpb1 funnel domain ...Sigma factor-binding protein Crl monomer / Sigma factor-binding transcriptional regulator Crl / Sigma factor-binding protein Crl superfamily / Sigma factor-binding transcriptional regulator Crl / RNA polymerase sigma factor RpoS / RNA polymerase II, Rpb2 subunit, wall domain / TATA-Binding Protein / RNA polymerase subunit, RPB6/omega / Eukaryotic RPB6 RNA polymerase subunit / RNA polymerase Rpb1 funnel domain / RNA polymerase, RBP11-like subunit / Sigma-70 factors family signature 1. / 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 Alpha Subunit; Chain A, domain 2 / RNA polymerase sigma-70 / 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 Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, RBP11-like subunit / 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
DNA / DNA (> 10) / Sigma factor-binding protein Crl / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit beta ...DNA / DNA (> 10) / Sigma factor-binding protein Crl / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit beta / Sigma factor-binding protein Crl / RNA polymerase sigma factor RpoS
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Salmonella typhimurium (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.26 Å
AuthorsJaramillo Cartagena, A. / Darst, S.A. / Campbell, E.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 basis for transcription activation by Crl through tethering of σ and RNA polymerase.
Authors: Alexis Jaramillo Cartagena / Amy B Banta / Nikhil Sathyan / Wilma Ross / Richard L Gourse / Elizabeth A Campbell / Seth A Darst /
Abstract: In bacteria, a primary σ-factor associates with the core RNA polymerase (RNAP) to control most transcription initiation, while alternative σ-factors are used to coordinate expression of additional ...In bacteria, a primary σ-factor associates with the core RNA polymerase (RNAP) to control most transcription initiation, while alternative σ-factors are used to coordinate expression of additional regulons in response to environmental conditions. Many alternative σ-factors are negatively regulated by anti-σ-factors. In , , and many other γ-proteobacteria, the transcription factor Crl positively regulates the alternative σ-regulon by promoting the association of σ with RNAP without interacting with promoter DNA. The molecular mechanism for Crl activity is unknown. Here, we determined a single-particle cryo-electron microscopy structure of Crl-σ-RNAP in an open promoter complex with a σ-regulon promoter. In addition to previously predicted interactions between Crl and domain 2 of σ (σ), the structure, along with -benzoylphenylalanine cross-linking, reveals that Crl interacts with a structural element of the RNAP β'-subunit that we call the β'-clamp-toe (β'CT). Deletion of the β'CT decreases activation by Crl without affecting basal transcription, highlighting the functional importance of the Crl-β'CT interaction. We conclude that Crl activates σ-dependent transcription in part through stabilizing σ-RNAP by tethering σ and the β'CT. We propose that Crl, and other transcription activators that may use similar mechanisms, be designated σ-activators.
History
DepositionApr 18, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 28, 2019Provider: repository / Type: Initial release
Revision 1.1Sep 18, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Oct 2, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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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 RpoS
J: Sigma factor-binding protein Crl
T: Template DNA strand
N: Non-template DNA strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)464,37612
Polymers464,2219
Non-polymers1553
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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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
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: rpoA, Z4665, ECs4160 / Production host: Escherichia coli (E. coli)
References: UniProt: P0A7Z6, UniProt: P0A7Z4*PLUS, 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, Z5560, ECs4910 / Production host: Escherichia coli (E. coli)
References: UniProt: P0A8V4, UniProt: P0A8V2*PLUS, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 155366.781 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 (E. coli) / 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, Z5075, ECs4524 / Production host: Escherichia coli (E. coli)
References: UniProt: P0A802, UniProt: P0A800*PLUS, DNA-directed RNA polymerase

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Protein , 2 types, 2 molecules FJ

#5: Protein RNA polymerase sigma factor RpoS / Sigma S / Sigma-38


Mass: 38069.770 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella typhimurium (bacteria) / Gene: rpoS, C2273_13580 / Production host: Escherichia coli (E. coli) / References: UniProt: S5ZIY8
#6: Protein Sigma factor-binding protein Crl


Mass: 16109.327 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella typhimurium (bacteria) / Gene: crl / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0F7J6B7, UniProt: Q7CR52*PLUS

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DNA chain , 2 types, 2 molecules TN

#7: DNA chain Template DNA strand


Mass: 20325.027 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#8: DNA chain Non-template DNA strand


Mass: 20361.086 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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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: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: CryoEM structure of SigmaS-transcription initiation complex with activator Crl
Type: COMPLEX / Entity ID: #1-#8 / Source: RECOMBINANT
Molecular weightValue: 0.523783 MDa / Experimental value: YES
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMK-GlutamateK-Glutamate1
250 mMtris(hydroxymethyl)aminomethaneTris-HCl1
310 mMMagnesium chlorideMgCl21
410 mMDithiothreitolDTT1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3 4C
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 22500 X
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 1.42 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1Gautomatchparticle selection
2SerialEMimage acquisition
4RELION3CTF correction
10RELION3initial Euler assignment
11cryoSPARC2final Euler assignment
13cryoSPARC23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 658000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.26 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 292000 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT

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