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- PDB-7q4u: Cryo-EM structure of Mycobacterium tuberculosis RNA polymerase ho... -

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Basic information

Entry
Database: PDB / ID: 7q4u
TitleCryo-EM structure of Mycobacterium tuberculosis RNA polymerase holoenzyme octamer comprising sigma factor SigB
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • RNA polymerase sigma factor SigB
KeywordsTRANSCRIPTION / DNA-dependent RNA polymerase / alternative sigma
Function / homology
Function and homology information


RNA polymerase core enzyme binding / Antimicrobial action and antimicrobial resistance in Mtb / sigma factor activity / peptidoglycan-based cell wall / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat ...RNA polymerase core enzyme binding / Antimicrobial action and antimicrobial resistance in Mtb / sigma factor activity / peptidoglycan-based cell wall / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat / response to hypoxia / protein dimerization activity / response to xenobiotic stimulus / response to antibiotic / DNA-templated transcription / positive regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Sigma-70 factors family signature 1. / 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 sigma-70 region 4 / Sigma-70, region 4 / RNA polymerase sigma-70 region 2 ...Sigma-70 factors family signature 1. / 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 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 / 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 / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb1, clamp domain superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 4 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 5 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / 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 / DNA-directed RNA polymerase, insert domain superfamily / 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 / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
RNA polymerase sigma factor SigB / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit alpha
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.39 Å
AuthorsBrodolin, K.
Funding support France, 1items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-16-CE11-0025 France
Citation
Journal: Nat Commun / Year: 2023
Title: Structural basis of the mycobacterial stress-response RNA polymerase auto-inhibition via oligomerization
Authors: Morichaud, Z. / Trapani, S. / Vishwakarma, R.K. / Chaloin, L. / Lionne, C. / Lai-Kee-Him, J. / Bron, P. / Brodolin, K.
#1: Journal: Biorxiv / Year: 2022
Title: Structural basis of the mycobacterial stress-response RNA polymerase auto-inhibition via oligomerization
Authors: Morichaud, Z. / Trapani, S. / Vishwakarma, R. / Chaloin, L. / Lionne, C. / Lai-Kee-Him, J. / Bron, P. / Brodolin, K.
History
DepositionNov 2, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 16, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 8, 2023Group: Database references / Refinement description
Category: citation / citation_author / pdbx_initial_refinement_model
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _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

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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 SigB
G: DNA-directed RNA polymerase subunit alpha
H: DNA-directed RNA polymerase subunit alpha
I: DNA-directed RNA polymerase subunit beta
J: DNA-directed RNA polymerase subunit beta'
K: DNA-directed RNA polymerase subunit omega
L: RNA polymerase sigma factor SigB
M: DNA-directed RNA polymerase subunit alpha
N: DNA-directed RNA polymerase subunit alpha
O: DNA-directed RNA polymerase subunit beta
P: DNA-directed RNA polymerase subunit beta'
Q: DNA-directed RNA polymerase subunit omega
R: RNA polymerase sigma factor SigB
S: DNA-directed RNA polymerase subunit alpha
T: DNA-directed RNA polymerase subunit alpha
V: DNA-directed RNA polymerase subunit beta
W: DNA-directed RNA polymerase subunit beta'
X: DNA-directed RNA polymerase subunit omega
Y: RNA polymerase sigma factor SigB
Z: DNA-directed RNA polymerase subunit alpha
AA: DNA-directed RNA polymerase subunit alpha
BA: DNA-directed RNA polymerase subunit beta
CA: DNA-directed RNA polymerase subunit beta'
DA: DNA-directed RNA polymerase subunit omega
EA: RNA polymerase sigma factor SigB
FA: DNA-directed RNA polymerase subunit alpha
GA: DNA-directed RNA polymerase subunit alpha
HA: DNA-directed RNA polymerase subunit beta
IA: DNA-directed RNA polymerase subunit beta'
JA: DNA-directed RNA polymerase subunit omega
KA: RNA polymerase sigma factor SigB
LA: DNA-directed RNA polymerase subunit alpha
MA: DNA-directed RNA polymerase subunit alpha
NA: DNA-directed RNA polymerase subunit beta
OA: DNA-directed RNA polymerase subunit beta'
PA: DNA-directed RNA polymerase subunit omega
QA: RNA polymerase sigma factor SigB
RA: DNA-directed RNA polymerase subunit alpha
SA: DNA-directed RNA polymerase subunit alpha
TA: DNA-directed RNA polymerase subunit beta
UA: DNA-directed RNA polymerase subunit beta'
VA: DNA-directed RNA polymerase subunit omega
WA: RNA polymerase sigma factor SigB
hetero molecules


Theoretical massNumber of molelcules
Total (without water)3,224,88372
Polymers3,223,64248
Non-polymers1,24124
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, equilibrium centrifugation
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA-directed RNA polymerase subunit ... , 4 types, 40 molecules ABGHMNSTZAAFAGALAMARASACIOVBAHANATADJPWCAIA...

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


Mass: 37745.328 Da / Num. of mol.: 16
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Strain: ATCC 25618 / H37Rv / Gene: rpoA, Rv3457c, MTCY13E12.10c / Plasmid: pMR4 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P9WGZ1, DNA-directed RNA polymerase
#2: Protein
DNA-directed RNA polymerase subunit beta / Polymerase / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 129602.344 Da / Num. of mol.: 8 / Mutation: L2E3G4C5 -> V
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Strain: ATCC 25618 / H37Rv / Gene: rpoB, Rv0667, MTCI376.08c / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P9WGY9, DNA-directed RNA polymerase
#3: Protein
DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 147438.344 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Strain: ATCC 25618 / H37Rv / Gene: rpoC, Rv0668, MTCI376.07c / Plasmid: pMR4 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P9WGY7, DNA-directed RNA polymerase
#4: Protein
DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11851.140 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Strain: ATCC 25618 / H37Rv / Gene: rpoZ, Rv1390, MTCY21B4.07 / Plasmid: pMR4 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P9WGY5, DNA-directed RNA polymerase

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Protein , 1 types, 8 molecules FLRYEAKAQAWA

#5: Protein
RNA polymerase sigma factor SigB


Mass: 38572.773 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Strain: ATCC 25618 / H37Rv / Gene: sigB, mysB, Rv2710 / Plasmid: pET28a / Details (production host): derivative containing Rv2710 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P9WGI5

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Non-polymers , 2 types, 24 molecules

#6: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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: RNA polymerase holoenzyme with sigma factor sigB / Type: COMPLEX
Details: RNA polymerase holoenzyme assembled from individually expressed RNA polymerase core and sigma factor sigB
Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightValue: 3.2 MDa / Experimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pMR4, pET28a
Buffer solutionpH: 7.9
SpecimenConc.: 1.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
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 recordingElectron dose: 49.6 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 3064

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
4GctfCTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 254380
SymmetryPoint symmetry: D4 (2x4 fold dihedral)
3D reconstructionResolution: 4.39 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 115457 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 7PP4

7pp4

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