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- PDB-9fjr: Cryo-EM structure of Mycobacterium tuberculosis sigma-B RNA polym... -

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Basic information

Entry
Database: PDB / ID: 9fjr
TitleCryo-EM structure of Mycobacterium tuberculosis sigma-B RNA polymerase bound to -10 promoter element ssDNA oligo - sigma-B docked conformation
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • DNA (17-MER)
  • RNA polymerase sigma factor SigB
KeywordsTRANSCRIPTION / RNA polymerase / sigma factor / SigB / stress response / -10 promoter element / promoter recognition / tuberculosis
Function / homology
Function and homology information


RNA polymerase core enzyme binding / Antimicrobial action and antimicrobial resistance in Mtb / sigma factor activity / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / DNA-directed RNA polymerase complex / peptidoglycan-based cell wall / DNA-templated transcription initiation / ribonucleoside binding / : ...RNA polymerase core enzyme binding / Antimicrobial action and antimicrobial resistance in Mtb / sigma factor activity / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / DNA-directed RNA polymerase complex / peptidoglycan-based cell wall / DNA-templated transcription initiation / ribonucleoside binding / : / : / : / : / : / : / DNA-directed RNA polymerase / response to heat / response to hypoxia / protein dimerization activity / response to xenobiotic stimulus / response to antibiotic / 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 ...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 / 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 / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / 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 H37Rv (bacteria)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.43 Å
AuthorsBrodolin, K. / Blaise, M.
Funding support France, 3items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-20-CE44-0020-01 France
Agence Nationale de la Recherche (ANR)ANR-16-CE11-0025 France
Instruct-ERIC Center (Strasbourg Centre)19348 France
Citation
Journal: Nucleic Acids Res / Year: 2025
Title: Single-stranded DNA drives σ subunit loading onto mycobacterial RNA polymerase to unlock initiation-competent conformations.
Authors: Rishi Kishore Vishwakarma / Nils Marechal / Zakia Morichaud / Mickaël Blaise / Emmanuel Margeat / Konstantin Brodolin /
Abstract: Initiation of transcription requires the formation of the "open" promoter complex (RPo). For this, the σ subunit of bacterial RNA polymerase (RNAP) binds to the nontemplate strand of the -10 element ...Initiation of transcription requires the formation of the "open" promoter complex (RPo). For this, the σ subunit of bacterial RNA polymerase (RNAP) binds to the nontemplate strand of the -10 element sequence of promoters and nucleates DNA unwinding. This is accompanied by a cascade of conformational changes on RNAP, the exact mechanics of which remains elusive. Here, using single-molecule Förster resonance energy transfer and cryo-electron microscopy, we explored the conformational landscape of RNAP from the human pathogen Mycobacterium tuberculosis upon binding to a single-stranded DNA (ssDNA) fragment that includes the -10 element sequence (-10 ssDNA). We found that like the transcription activator RNAP-binding protein A, -10 ssDNA induced σ subunit loading onto the DNA/RNA channels of RNAP. This triggered RNAP clamp closure and unswiveling that are required for RPo formation and RNA synthesis initiation. Our results reveal a mechanism of ssDNA-guided RNAP maturation and identify the σ subunit as a regulator of RNAP conformational dynamics.
#1: Journal: Nat Commun / Year: 2023
Title: Structural basis of the mycobacterial stress-response RNA polymerase auto-inhibition via oligomerization.
Authors: Zakia Morichaud / Stefano Trapani / Rishi K Vishwakarma / Laurent Chaloin / Corinne Lionne / Joséphine Lai-Kee-Him / Patrick Bron / Konstantin Brodolin /
Abstract: Self-assembly of macromolecules into higher-order symmetric structures is fundamental for the regulation of biological processes. Higher-order symmetric structure self-assembly by the gene expression ...Self-assembly of macromolecules into higher-order symmetric structures is fundamental for the regulation of biological processes. Higher-order symmetric structure self-assembly by the gene expression machinery, such as bacterial DNA-dependent RNA polymerase (RNAP), has never been reported before. Here, we show that the stress-response σ factor from the human pathogen, Mycobacterium tuberculosis, induces the RNAP holoenzyme oligomerization into a supramolecular complex composed of eight RNAP units. Cryo-electron microscopy revealed a pseudo-symmetric structure of the RNAP octamer in which RNAP protomers are captured in an auto-inhibited state and display an open-clamp conformation. The structure shows that σ is sequestered by the RNAP flap and clamp domains. The transcriptional activator RbpA prevented octamer formation by promoting the initiation-competent RNAP conformation. Our results reveal that a non-conserved region of σ is an allosteric controller of transcription initiation and demonstrate how basal transcription factors can regulate gene expression by modulating the RNAP holoenzyme assembly and hibernation.
#2: Journal: Nucleic Acids Res / Year: 2018
Title: RbpA relaxes promoter selectivity of M. tuberculosis RNA polymerase.
Authors: Ayyappasamy Sudalaiyadum Perumal / Rishi Kishore Vishwakarma / Yangbo Hu / Zakia Morichaud / Konstantin Brodolin /
Abstract: The transcriptional activator RbpA associates with Mycobacterium tuberculosis RNA polymerase (MtbRNAP) during transcription initiation, and stimulates formation of the MtbRNAP-promoter open complex ...The transcriptional activator RbpA associates with Mycobacterium tuberculosis RNA polymerase (MtbRNAP) during transcription initiation, and stimulates formation of the MtbRNAP-promoter open complex (RPo). Here, we explored the influence of promoter motifs on RbpA-mediated activation of MtbRNAP containing the stress-response σB subunit. We show that both the 'extended -10' promoter motif (T-17G-16T-15G-14) and RbpA stabilized RPo and allowed promoter opening at suboptimal temperatures. Furthermore, in the presence of the T-17G-16T-15G-14 motif, RbpA was dispensable for RNA synthesis initiation, while exerting a stabilization effect on RPo. On the other hand, RbpA compensated for the lack of sequence-specific interactions of domains 3 and 4 of σB with the extended -10 and the -35 motifs, respectively. Mutations of the positively charged residues K73, K74 and R79 in RbpA basic linker (BL) had little effect on RPo formation, but affected MtbRNAP capacity for de novo transcription initiation. We propose that RbpA stimulates transcription by strengthening the non-specific interaction of the σ subunit with promoter DNA upstream of the -10 element, and by indirectly optimizing MtbRNAP interaction with initiation substrates. Consequently, RbpA renders MtbRNAP promiscuous in promoter selection, thus compensating for the weak conservation of the -35 motif in mycobacteria.
History
DepositionMay 31, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 7, 2025Provider: repository / Type: Initial release

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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
O: DNA (17-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)408,40810
Polymers408,2537
Non-polymers1553
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable, cross-linking, formaldehyde mediated DNA-protein cross-linking assay
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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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: 37745.328 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
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 / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 129602.344 Da / Num. of mol.: 1 / Mutation: L2E3G4C5I6 -> V
Source method: isolated from a genetically manipulated source
Details: he N-terminal resides, L2E3G4C5I6, are replaced by V
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: rpoB, Rv0667, MTCI376.08c / Plasmid: pMR4 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P9WGY9, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 147438.344 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: contains C-terminal 6xHis-tag
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
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 / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11851.140 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
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 / DNA chain , 2 types, 2 molecules fO

#5: Protein RNA polymerase sigma factor SigB


Mass: 38572.773 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Addition of twenty N-terminal residues (MGSSHHHHHHSSGLVPRGSH) including 6xHIS tag
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: sigB, mysB, Rv2710 / Plasmid: pET28a-sigB
Details (production host): pET28 derivative comprising Rv2710 gene
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P9WGI5
#6: DNA chain DNA (17-MER)


Mass: 5297.444 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: synthetic oligonucleotide / Source: (synth.) Escherichia coli (E. coli)

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Non-polymers , 2 types, 3 molecules

#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationN

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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 bound to -10 promoter element ssDNA oligo - sigB docked state
Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightValue: 0.406 MDa / Experimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pMR4, pET28-sigB
Buffer solutionpH: 7.9 / Details: 8 mM CHAPSO added before vitrificaion
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMpotassium chlorideKCl1
35 mMmagnesium chlorideMgCl21
42 mMdithiothreitolC4H10O2S21
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 291.15 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: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / Cs: 0.01 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.997 sec. / Electron dose: 55.735 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9202 / Details: images were collected in super-resolution mode

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Processing

EM software
IDNameVersionCategory
1Warpparticle selection
4cryoSPARC3.3CTF correction
7UCSF Chimeramodel fitting
8Coot0.9.1model fitting
10PHENIXmodel refinement
11cryoSPARC3.3initial Euler assignment
12cryoSPARC3.3final Euler assignment
13cryoSPARC3.3classification
14cryoSPARC3.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 695496
3D reconstructionResolution: 3.43 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 72799 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building

3D fitting-ID: 1

IDPDB-IDAccession codeInitial refinement model-IDSource nameTypeChain-IDChain residue range
17PP4

7pp4

7PP41PDBexperimental model
2AlphaFoldin silico modelf159-323
RefinementCross valid method: NONE

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