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- PDB-8hih: Cryo-EM structure of Mycobacterium tuberculosis transcription ini... -

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Basic information

Entry
Database: PDB / ID: 8hih
TitleCryo-EM structure of Mycobacterium tuberculosis transcription initiation complex with transcription factor GlnR
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • Non-template strand DNA of amtB promoter
  • RNA polymerase sigma factor SigA
  • Template strand DNA of amtB promoter
  • Transcriptional regulatory protein
KeywordsTRANSCRIPTION/DNA / transcription factor / TRANSCRIPTION-DNA complex
Function / homology
Function and homology information


response to water / Antimicrobial action and antimicrobial resistance in Mtb / phosphorelay response regulator activity / sigma factor activity / nitrate assimilation / DNA-directed RNA polymerase complex / peptidoglycan-based cell wall / DNA-templated transcription initiation / protein-DNA complex / ribonucleoside binding ...response to water / Antimicrobial action and antimicrobial resistance in Mtb / phosphorelay response regulator activity / sigma factor activity / nitrate assimilation / DNA-directed RNA polymerase complex / peptidoglycan-based cell wall / DNA-templated transcription initiation / protein-DNA complex / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / transcription cis-regulatory region binding / response to antibiotic / DNA-templated transcription / regulation of DNA-templated transcription / positive regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / plasma membrane / cytoplasm / cytosol
Similarity search - Function
: / Transcription regulator GlnR, N-terminal domain / OmpR/PhoB-type DNA-binding domain profile. / OmpR/PhoB-type DNA-binding domain / Transcriptional regulatory protein, C terminal / Transcriptional regulatory protein, C terminal / Transcriptional regulatory protein WalR-like / Signal transduction response regulator, C-terminal effector / Sigma-70 factors family signature 1. / RNA polymerase sigma factor RpoD, C-terminal ...: / Transcription regulator GlnR, N-terminal domain / OmpR/PhoB-type DNA-binding domain profile. / OmpR/PhoB-type DNA-binding domain / Transcriptional regulatory protein, C terminal / Transcriptional regulatory protein, C terminal / Transcriptional regulatory protein WalR-like / Signal transduction response regulator, C-terminal effector / Sigma-70 factors family signature 1. / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / 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 / CheY-like superfamily / 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 Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / 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, beta subunit, protrusion / RNA polymerase beta subunit / 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 / 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
DNA / DNA (> 10) / DNA (> 100) / Transcriptional regulatory protein / RNA polymerase sigma factor SigA / 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)
Mycobacterium tuberculosis H37Rv (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.66 Å
AuthorsLin, W. / Shi, J. / Xu, J.C.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)81903526 China
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Structural insights into the transcription activation mechanism of the global regulator GlnR from actinobacteria.
Authors: Jing Shi / Zhenzhen Feng / Juncao Xu / Fangfang Li / Yuqiong Zhang / Aijia Wen / Fulin Wang / Qian Song / Lu Wang / Hong Cui / Shujuan Tong / Peiying Chen / Yejin Zhu / Guoping Zhao / Shuang ...Authors: Jing Shi / Zhenzhen Feng / Juncao Xu / Fangfang Li / Yuqiong Zhang / Aijia Wen / Fulin Wang / Qian Song / Lu Wang / Hong Cui / Shujuan Tong / Peiying Chen / Yejin Zhu / Guoping Zhao / Shuang Wang / Yu Feng / Wei Lin /
Abstract: In actinobacteria, an OmpR/PhoB subfamily protein called GlnR acts as an orphan response regulator and globally coordinates the expression of genes responsible for nitrogen, carbon, and phosphate ...In actinobacteria, an OmpR/PhoB subfamily protein called GlnR acts as an orphan response regulator and globally coordinates the expression of genes responsible for nitrogen, carbon, and phosphate metabolism in actinobacteria. Although many researchers have attempted to elucidate the mechanisms of GlnR-dependent transcription activation, progress is impeded by lacking of an overall structure of GlnR-dependent transcription activation complex (GlnR-TAC). Here, we report a co-crystal structure of the C-terminal DNA-binding domain of GlnR (GlnR_DBD) in complex with its regulatory -element DNA and a cryo-EM structure of GlnR-TAC which comprises RNA polymerase, GlnR, and a promoter containing four well-characterized conserved GlnR binding sites. These structures illustrate how four GlnR protomers coordinate to engage promoter DNA in a head-to-tail manner, with four N-terminal receiver domains of GlnR (GlnR-RECs) bridging GlnR_DBDs and the RNAP core enzyme. Structural analysis also unravels that GlnR-TAC is stabilized by complex protein-protein interactions between GlnR and the conserved β flap, σR4, αCTD, and αNTD domains of RNAP, which are further confirmed by our biochemical assays. Taken together, these results reveal a global transcription activation mechanism for the master regulator GlnR and other OmpR/PhoB subfamily proteins and present a unique mode of bacterial transcription regulation.
History
DepositionNov 20, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 7, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 3, 2024Group: Data collection / Other
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_database_status
Item: _em_admin.last_update / _pdbx_database_status.pdb_format_compatible

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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 SigA
G: DNA-directed RNA polymerase subunit alpha
K: Non-template strand DNA of amtB promoter
L: Template strand DNA of amtB promoter
O: Transcriptional regulatory protein
P: Transcriptional regulatory protein
N: Transcriptional regulatory protein
Q: Transcriptional regulatory protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)638,07616
Polymers637,92113
Non-polymers1553
Water00
1


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

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Components

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DNA-directed RNA polymerase subunit ... , 4 types, 6 molecules ABGCDE

#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.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoA / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / 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: 130018.828 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoB / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / 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: 146968.969 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoC / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / 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)
Strain: H37Rv / Gene: rpoZ / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P9WGY5, DNA-directed RNA polymerase

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Protein , 2 types, 5 molecules FOPNQ

#5: Protein RNA polymerase sigma factor SigA / Sigma-A


Mass: 57877.160 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Strain: H37Rv / Gene: sigA / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P9WGI1
#8: Protein
Transcriptional regulatory protein


Mass: 27631.398 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Strain: H37Rv / Gene: Rv0818 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: O53830

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DNA chain , 2 types, 2 molecules KL

#6: DNA chain Non-template strand DNA of amtB promoter


Mass: 33588.414 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mycobacterium tuberculosis (bacteria)
#7: DNA chain Template strand DNA of amtB promoter


Mass: 33854.512 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mycobacterium tuberculosis (bacteria)

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Non-polymers , 2 types, 3 molecules

#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#10: 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

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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: Mycobacterium tuberculosis transcription initiation complex with transcription factor GlnR
Type: COMPLEX / Entity ID: #1-#8 / Source: RECOMBINANT
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
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 FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 57 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 202298
Details: The higher resolution region than depositor's reported resolution of the data is reflected by CTF correction or temperature factor correction.
Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00532707
ELECTRON MICROSCOPYf_angle_d0.68144964
ELECTRON MICROSCOPYf_dihedral_angle_d20.4845612
ELECTRON MICROSCOPYf_chiral_restr0.0445126
ELECTRON MICROSCOPYf_plane_restr0.0065340

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