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- PDB-6k4y: CryoEM structure of sigma appropriation complex -

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Basic information

Entry
Database: PDB / ID: 6k4y
TitleCryoEM structure of sigma appropriation complex
Components
  • (DNA (60-MER)) x 2
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • 10 kDa anti-sigma factor
  • Middle transcription regulatory protein motA
  • RNA polymerase sigma factor RpoD
KeywordsTRANSCRIPTION / RNA polymerase / sigma appropriation / transcription activation / promoter
Function / homology
Function and homology information


RNA polymerase complex / transcription initiation from bacterial-type RNA polymerase promoter / sigma factor activity / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein-containing complex assembly / response to heat / protein dimerization activity / transcription, DNA-templated ...RNA polymerase complex / transcription initiation from bacterial-type RNA polymerase promoter / sigma factor activity / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein-containing complex assembly / response to heat / protein dimerization activity / transcription, DNA-templated / response to antibiotic / regulation of transcription, DNA-templated / DNA-binding transcription factor activity / DNA binding / zinc ion binding / membrane / metal ion binding / cytosol / cytoplasm
Anti-Sigma Factor A / Bacteriophage T4, MotA, transcription regulator N-terminal / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / RPB6/omega subunit-like superfamily / RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase, beta subunit, external 1 domain / DNA-directed RNA polymerase, subunit 2 / Transcription regulator MotA, C-terminal / RNA polymerase Rpb2, OB-fold ...Anti-Sigma Factor A / Bacteriophage T4, MotA, transcription regulator N-terminal / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / RPB6/omega subunit-like superfamily / RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase, beta subunit, external 1 domain / DNA-directed RNA polymerase, subunit 2 / Transcription regulator MotA, C-terminal / RNA polymerase Rpb2, OB-fold / Anti-Sigma Factor A superfamily / RNA polymerase sigma-70 like domain / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like / RNA polymerase sigma factor RpoD, C-terminal / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase subunit, RPB6/omega / DNA-directed RNA polymerase, alpha subunit / Transcription regulator MotA, C-terminal domain superfamily / RNA polymerase, RBP11-like subunit / DNA-directed RNA polymerase, insert domain / RNA polymerase Rpb6 / Sigma-70, non-essential region / Sigma-70, region 4 / Sigma-70 region 2 / Sigma-70 region 3 / Sigma-70 factor, region 1.1 / Bacterial RNA polymerase, alpha chain C terminal domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase Rpb3/RpoA insert domain / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb2, domain 6 / Sigma-70 factor, region 1.2 / Sigma-70 factor, region 1.1 superfamily / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase, alpha subunit, C-terminal / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase sigma-70 / RNA polymerase, alpha subunit / RNA polymerase beta subunit / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 4 / RNA polymerase, subunit omega/K/RPB6 / Anti-Sigma Factor A / Transcription factor MotA, activation domain / Bacteriophage T4 MotA, C-terminal / RNA polymerase beta subunit external 1 domain / Sigma-70 factors family signature 1. / Sigma-70 factors family signature 2. / RNA polymerases beta chain signature. / DNA-directed RNA polymerase beta subunit, bacterial-type / DNA-directed RNA polymerase, omega subunit / RNA polymerase, N-terminal / RNA polymerase sigma-70 region 3 / RNA polymerase sigma-70 region 1.2 / RNA polymerase Rpb2, domain 3 / RNA polymerase, beta subunit, protrusion / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 7 / RNA polymerase sigma factor 70, non-essential domain / RNA polymerase sigma-70 region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb1, domain 3 / RNA polymerase sigma factor 70, region 1.1 / RNA polymerase, beta subunit, conserved site / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 1
RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / Middle transcription regulatory protein motA / 10 kDa anti-sigma factor
Biological speciesEscherichia coli K-12 (bacteria)
Enterobacteria phage T4 (bacteriophage)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.79 Å
AuthorsShi, J. / Wen, A. / Feng, Y.
Funding support China, 1items
OrganizationGrant numberCountry
Ministry of Science and Technology (China)2018YFA0507800 China
CitationJournal: Nucleic Acids Res. / Year: 2019
Title: Structural basis of σ appropriation.
Authors: Jing Shi / Aijia Wen / Minxing Zhao / Linlin You / Yu Zhang / Yu Feng /
Abstract: Bacteriophage T4 middle promoters are activated through a process called σ appropriation, which requires the concerted effort of two T4-encoded transcription factors: AsiA and MotA. Despite ...Bacteriophage T4 middle promoters are activated through a process called σ appropriation, which requires the concerted effort of two T4-encoded transcription factors: AsiA and MotA. Despite extensive biochemical and genetic analyses, puzzle remains, in part, because of a lack of precise structural information for σ appropriation complex. Here, we report a single-particle cryo-electron microscopy (cryo-EM) structure of an intact σ appropriation complex, comprising AsiA, MotA, Escherichia coli RNA polymerase (RNAP), σ70 and a T4 middle promoter. As expected, AsiA binds to and remodels σ region 4 to prevent its contact with host promoters. Unexpectedly, AsiA undergoes a large conformational change, takes over the job of σ region 4 and provides an anchor point for the upstream double-stranded DNA. Because σ region 4 is conserved among bacteria, other transcription factors may use the same strategy to alter the landscape of transcription immediately. Together, the structure provides a foundation for understanding σ appropriation and transcription activation.
Validation Report
SummaryFull reportAbout validation report
History
DepositionMay 27, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 7, 2019Provider: repository / Type: Initial release

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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 RpoD
I: 10 kDa anti-sigma factor
M: Middle transcription regulatory protein motA
N: DNA (60-MER)
T: DNA (60-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)533,59313
Polymers533,43810
Non-polymers1553
Water0
1


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/peptide DNA-directed RNA polymerase subunit alpha / Polymerase / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 36558.680 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K-12 / Gene: rpoA / Production host: Escherichia coli (E. coli) / References: UniProt: P0A7Z4, DNA-directed RNA polymerase
#2: Protein/peptide DNA-directed RNA polymerase subunit beta / Polymerase / 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 K-12 (bacteria) / Strain: K-12 / Gene: rpoB / Production host: Escherichia coli (E. coli) / References: UniProt: P0A8V2, DNA-directed RNA polymerase
#3: Protein/peptide DNA-directed RNA polymerase subunit beta' / Polymerase / 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 K-12 (bacteria) / Strain: K-12 / Gene: rpoC / Production host: Escherichia coli (E. coli) / References: UniProt: P0A8T7, DNA-directed RNA polymerase
#4: Protein/peptide DNA-directed RNA polymerase subunit omega / Polymerase / 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 K-12 (bacteria) / Strain: K-12 / Gene: rpoZ / Production host: Escherichia coli (E. coli) / References: UniProt: P0A800, DNA-directed RNA polymerase

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Protein/peptide , 3 types, 3 molecules FIM

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


Mass: 70352.242 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K-12 / Gene: rpoD / Production host: Escherichia coli (E. coli) / References: UniProt: P00579
#6: Protein/peptide 10 kDa anti-sigma factor / 10 kDa RNA polymerase-associated protein / Audrey Stevens' inhibitor


Mass: 12775.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterobacteria phage T4 (bacteriophage)
Gene: asiA / Production host: Escherichia coli (E. coli) / References: UniProt: P32267
#7: Protein/peptide Middle transcription regulatory protein motA


Mass: 23608.334 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterobacteria phage T4 (bacteriophage)
Gene: motA / Production host: Escherichia coli (E. coli) / References: UniProt: P22915

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DNA chain , 2 types, 2 molecules NT

#8: DNA chain DNA (60-MER)


Mass: 18642.982 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Enterobacteria phage T4 (bacteriophage)
#9: DNA chain DNA (60-MER)


Mass: 18504.877 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Enterobacteria phage T4 (bacteriophage)

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Non-polymers , 2 types, 3 molecules

#10: Chemical ChemComp-MG / MAGNESIUM ION


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


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

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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: sigma appropriation complex / Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6, 7, 8, 9 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)

Entity assembly-ID: 1

IDOrganismNcbi tax-ID
1Escherichia coli K-12 (bacteria)83333
2Escherichia virus T410665
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.9
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy
Image recordingAverage exposure time: 12 sec. / Electron dose: 56 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.79 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 105108 / Symmetry type: POINT

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