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- PDB-7f75: Cryo-EM structure of Spx-dependent transcription activation complex -

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Basic information

Entry
Database: PDB / ID: 7f75
TitleCryo-EM structure of Spx-dependent transcription activation complex
Components
  • (DNA-directed RNA polymerase subunit ...) x 6
  • (trxA promoter DNA- ...) x 2
  • RNA polymerase sigma factor SigA
  • transcriptional regulator Spx
KeywordsTRANSCRIPTION/DNA / Complex / transcription activator / stress / oxidated form / TRANSCRIPTION / TRANSCRIPTION-DNA complex
Function / homology
Function and homology information


nucleoid / sigma factor activity / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / protein dimerization activity ...nucleoid / sigma factor activity / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / protein dimerization activity / response to antibiotic / DNA-templated transcription / regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm
Similarity search - Function
RNA polymerase epsilon subunit / RNA polymerase epsilon subunit / DNA-directed RNA polymerase subunit delta / DNA-directed RNA polymerase subunit delta, N-terminal domain superfamily / ASXL, HARE-HTH domain / HB1, ASXL, restriction endonuclease HTH domain / HARE-type HTH domain profile. / RNA polymerase sigma factor 70, region 1.1 / Sigma-70 factor, region 1.1 superfamily / Sigma-70 factor, region 1.1 ...RNA polymerase epsilon subunit / RNA polymerase epsilon subunit / DNA-directed RNA polymerase subunit delta / DNA-directed RNA polymerase subunit delta, N-terminal domain superfamily / ASXL, HARE-HTH domain / HB1, ASXL, restriction endonuclease HTH domain / HARE-type HTH domain profile. / RNA polymerase sigma factor 70, region 1.1 / Sigma-70 factor, region 1.1 superfamily / Sigma-70 factor, region 1.1 / 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 / DNA-directed RNA polymerase, omega subunit / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / 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 beta subunit, bacterial-type / DNA-directed RNA polymerase, alpha subunit / 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 Rpb2, domain 2 / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb2, domain 2 / RNA polymerase I subunit A N-terminus / 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 / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA-directed RNA polymerase subunit epsilon / DNA-directed RNA polymerase subunit omega / RNA polymerase sigma factor SigA / DNA-directed RNA polymerase subunit delta / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit beta'
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsLin, W. / Feng, Y. / Shi, J.
Funding support1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)
CitationJournal: Nucleic Acids Res / Year: 2021
Title: Structural basis of transcription activation by the global regulator Spx.
Authors: Jing Shi / Fangfang Li / Aijia Wen / Libing Yu / Lu Wang / Fulin Wang / Yuanling Jin / Sha Jin / Yu Feng / Wei Lin /
Abstract: Spx is a global transcriptional regulator in Gram-positive bacteria and has been inferred to efficiently activate transcription upon oxidative stress by engaging RNA polymerase (RNAP) and promoter ...Spx is a global transcriptional regulator in Gram-positive bacteria and has been inferred to efficiently activate transcription upon oxidative stress by engaging RNA polymerase (RNAP) and promoter DNA. However, the precise mechanism by which it interacts with RNAP and promoter DNA to initiate transcription remains obscure. Here, we report the cryo-EM structure of an intact Spx-dependent transcription activation complex (Spx-TAC) from Bacillus subtilis at 4.2 Å resolution. The structure traps Spx in an active conformation and defines key interactions accounting for Spx-dependent transcription activation. Strikingly, an oxidized Spx monomer engages RNAP by simultaneously interacting with the C-terminal domain of RNAP alpha subunit (αCTD) and σA. The interface between Spx and αCTD is distinct from those previously reported activators, indicating αCTD as a multiple target for the interaction between RNAP and various transcription activators. Notably, Spx specifically wraps the conserved -44 element of promoter DNA, thereby stabilizing Spx-TAC. Besides, Spx interacts extensively with σA through three different interfaces and promotes Spx-dependent transcription activation. Together, our structural and biochemical results provide a novel mechanistic framework for the regulation of bacterial transcription activation and shed new light on the physiological roles of the global Spx-family transcription factors.
History
DepositionJun 28, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 13, 2021Provider: repository / Type: Initial release
Revision 1.1Feb 16, 2022Group: 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 1.2Nov 13, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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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 SigA
H: DNA-directed RNA polymerase subunit epsilon
G: transcriptional regulator Spx
I: DNA-directed RNA polymerase subunit alpha
J: trxA promoter DNA-Non template strand
K: trxA promoter DNA-template strand
L: DNA-directed RNA polymerase subunit delta
hetero molecules


Theoretical massNumber of molelcules
Total (without water)510,06815
Polymers509,91312
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 ... , 6 types, 8 molecules ABICDEHL

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


Mass: 34842.387 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Bacillus subtilis (bacteria) / References: UniProt: P20429, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 133847.938 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bacillus subtilis (bacteria) / References: UniProt: P37870, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 134444.953 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bacillus subtilis (bacteria) / References: UniProt: P37871, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega 2 subunit / Transcriptase subunit omega


Mass: 7766.921 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bacillus subtilis (bacteria) / References: UniProt: O35011, DNA-directed RNA polymerase
#6: Protein DNA-directed RNA polymerase subunit epsilon / RNA polymerase auxiliary subunit epsilon subunit / RNAP epsilon subunit / DNA-directed RNA ...RNA polymerase auxiliary subunit epsilon subunit / RNAP epsilon subunit / DNA-directed RNA polymerase subunit omega 1 / RNA polymerase epsilon subunit / RNA polymerase omega 1 subunit / Transcriptase subunit epsilon / Transcriptase subunit omega 1


Mass: 8263.358 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bacillus subtilis (bacteria) / References: UniProt: O31718, DNA-directed RNA polymerase
#10: Protein DNA-directed RNA polymerase subunit delta / RNAP delta factor


Mass: 20417.891 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bacillus subtilis (bacteria) / References: UniProt: P12464

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Protein , 2 types, 2 molecules FG

#5: Protein RNA polymerase sigma factor SigA / Sigma-43 / Sigma-A / RNA polymerase sigma subunit


Mass: 43007.406 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bacillus subtilis (bacteria) / References: UniProt: P06224
#7: Protein transcriptional regulator Spx


Mass: 15547.901 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bacillus subtilis (bacteria)

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TrxA promoter DNA- ... , 2 types, 2 molecules JK

#8: DNA chain trxA promoter DNA-Non template strand


Mass: 21202.631 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Bacillus subtilis (bacteria)
#9: DNA chain trxA promoter DNA-template strand


Mass: 20886.408 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Bacillus subtilis (bacteria)

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Non-polymers , 2 types, 3 molecules

#11: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#12: Chemical ChemComp-ZN / ZINC ION


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

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Details

Has ligand of interestY
Has protein modificationY
Sequence detailsThe reference sequence database of chain G is GenBank WP_106610844.1

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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

Component
IDNameTypeEntity IDParent-IDSource
1Bacillus subtilis Spx dependent transcription activation complexCOMPLEX#1-#100MULTIPLE SOURCES
2Spx-dependent transcription activation complexCOMPLEX#1-#7, #101NATURAL
3DNACOMPLEX#8-#91SYNTHETIC
Source (natural)Organism: Bacillus subtilis (bacteria)
Buffer solutionpH: 7.5
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
Image recordingElectron dose: 59 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM softwareName: RELION / Category: final Euler assignment
CTF correctionType: NONE
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 153870 / Algorithm: FOURIER SPACE / Symmetry type: POINT

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