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- PDB-8h3v: Cryo-EM structure of the full transcription activation complex Nt... -

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Basic information

Entry
Database: PDB / ID: 8h3v
TitleCryo-EM structure of the full transcription activation complex NtcA-NtcB-TAC
Components
  • (DNA (125-MER)) x 2
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 5
  • NtcA
  • NtcB
  • RNA polymerase sigma factor SigA
KeywordsTRANSCRIPTION / transcription activation complex / coordinated regulation
Function / homology
Function and homology information


sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / DNA-binding transcription factor activity / DNA-templated transcription / magnesium ion binding ...sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / DNA-binding transcription factor activity / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm
Similarity search - Function
RNA polymerase sigma factor, RpoD-like, cyanobacteria / Transcription regulator, NtcA / DNA-directed RNA polymerase, subunit gamma / DNA-directed RNA polymerase subunit RpoC1 / DNA-directed RNA polymerase, subunit beta'' / Transcription regulator HTH, Crp-type, conserved site / Crp-type HTH domain signature. / helix_turn_helix, cAMP Regulatory protein / Crp-type HTH domain profile. / Crp-like helix-turn-helix domain ...RNA polymerase sigma factor, RpoD-like, cyanobacteria / Transcription regulator, NtcA / DNA-directed RNA polymerase, subunit gamma / DNA-directed RNA polymerase subunit RpoC1 / DNA-directed RNA polymerase, subunit beta'' / Transcription regulator HTH, Crp-type, conserved site / Crp-type HTH domain signature. / helix_turn_helix, cAMP Regulatory protein / Crp-type HTH domain profile. / Crp-like helix-turn-helix domain / Crp-type HTH domain / LysR, substrate-binding / LysR substrate binding domain / LysR-type HTH domain profile. / Transcription regulator HTH, LysR / Bacterial regulatory helix-turn-helix protein, lysR family / 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 / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / DNA-directed RNA polymerase, omega subunit / 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 / RmlC-like jelly roll fold / 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 DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA (> 100) / Global nitrogen regulator / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit gamma / DNA-directed RNA polymerase subunit beta' / RNA polymerase sigma factor SigA / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit alpha / Nitrogen assimilation transcriptional activator
Similarity search - Component
Biological speciesAnabaena (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsHan, S.J. / Jiang, Y.L. / You, L.L. / Shen, L.Q. / Wu, X.X. / Yang, F. / Kong, W.W. / Chen, Z.P. / Zhang, Y. / Zhou, C.Z.
Funding support China, 4items
OrganizationGrant numberCountry
Chinese Academy of SciencesXDB37020301, XDA24020302 China
National Natural Science Foundation of China (NSFC)32171198 China
Ministry of Science and Technology (MoST, China)2018YFA090070 China
Chinese Academy of Sciences2020452 China
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: DNA looping mediates cooperative transcription activation.
Authors: Shu-Jing Han / Yong-Liang Jiang / Lin-Lin You / Li-Qiang Shen / Xiaoxian Wu / Feng Yang / Ning Cui / Wen-Wen Kong / Hui Sun / Ke Zhou / Hui-Chao Meng / Zhi-Peng Chen / Yuxing Chen / Yu Zhang / Cong-Zhao Zhou /
Abstract: Transcription factors respond to multilevel stimuli and co-occupy promoter regions of target genes to activate RNA polymerase (RNAP) in a cooperative manner. To decipher the molecular mechanism, here ...Transcription factors respond to multilevel stimuli and co-occupy promoter regions of target genes to activate RNA polymerase (RNAP) in a cooperative manner. To decipher the molecular mechanism, here we report two cryo-electron microscopy structures of Anabaena transcription activation complexes (TACs): NtcA-TAC composed of RNAP holoenzyme, promoter and a global activator NtcA, and NtcA-NtcB-TAC comprising an extra context-specific regulator, NtcB. Structural analysis showed that NtcA binding makes the promoter DNA bend by ∼50°, which facilitates RNAP to contact NtcB at the distal upstream NtcB box. The sequential binding of NtcA and NtcB induces looping back of promoter DNA towards RNAP, enabling the assembly of a fully activated TAC bound with two activators. Together with biochemical assays, we propose a 'DNA looping' mechanism of cooperative transcription activation in bacteria.
History
DepositionOct 9, 2022Deposition site: PDBJ / Processing site: RCSB
Revision 1.0Oct 4, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 17, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Feb 28, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
1: DNA (125-MER)
2: DNA (125-MER)
A: DNA-directed RNA polymerase subunit beta
B: DNA-directed RNA polymerase subunit beta'
C: DNA-directed RNA polymerase subunit alpha
D: DNA-directed RNA polymerase subunit alpha
E: DNA-directed RNA polymerase subunit gamma
F: DNA-directed RNA polymerase subunit omega
G: RNA polymerase sigma factor SigA
S: NtcB
T: NtcB
U: NtcB
V: NtcB
X: NtcA
Y: NtcA


Theoretical massNumber of molelcules
Total (without water)718,72915
Polymers718,72915
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA chain , 2 types, 2 molecules 12

#1: DNA chain DNA (125-MER)


Mass: 38723.902 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Anabaena (bacteria)
#2: DNA chain DNA (125-MER)


Mass: 38571.723 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Anabaena (bacteria)

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DNA-directed RNA polymerase subunit ... , 5 types, 6 molecules ABCDEF

#3: Protein DNA-directed RNA polymerase subunit beta / Polymerase / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 126781.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Anabaena (bacteria) / Strain: PCC 7120 / SAG 25.82 / UTEX 2576 / Gene: rpoB, alr1594 / Production host: Escherichia coli (E. coli) / References: UniProt: P22703, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 147137.484 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Anabaena (bacteria) / Strain: PCC 7120 / SAG 25.82 / UTEX 2576 / Gene: rpoC2, alr1596 / Production host: Escherichia coli (E. coli) / References: UniProt: P22705, DNA-directed RNA polymerase
#5: Protein DNA-directed RNA polymerase subunit alpha / Polymerase / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 26166.613 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Anabaena (bacteria) / Strain: PCC 7120 / SAG 25.82 / UTEX 2576 / Gene: rpoA, all4191 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8YPK3, DNA-directed RNA polymerase
#6: Protein DNA-directed RNA polymerase subunit gamma / Polymerase / RNAP subunit gamma / RNA polymerase subunit gamma / Transcriptase subunit gamma


Mass: 70653.055 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Anabaena (bacteria) / Strain: PCC 7120 / SAG 25.82 / UTEX 2576 / Gene: rpoC1, alr1595 / Production host: Escherichia coli (E. coli) / References: UniProt: P22704, DNA-directed RNA polymerase
#7: Protein DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 9143.490 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Anabaena (bacteria) / Strain: PCC 7120 / SAG 25.82 / UTEX 2576 / Gene: rpoZ, asr4648 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8YNB9, DNA-directed RNA polymerase

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Protein , 3 types, 7 molecules GSTUVXY

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


Mass: 45717.027 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Anabaena (bacteria) / Strain: PCC 7120 / SAG 25.82 / UTEX 2576 / Gene: sigA, rpoD, all5263 / Production host: Escherichia coli (E. coli) / References: UniProt: P26683
#9: Protein
NtcB


Mass: 34941.363 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Anabaena (bacteria) / Strain: PCC 7120 / SAG 25.82 / UTEX 2576 / Gene: ntcB / Production host: Escherichia coli (E. coli) / References: UniProt: Q9L3R4
#10: Protein NtcA / DNA-binding protein VF1 / Nitrogen-responsive regulatory protein


Mass: 24951.266 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Anabaena (bacteria) / Strain: PCC 7120 / SAG 25.82 / UTEX 2576 / Gene: ntcA, bifA, alr4392 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A4U6

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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: full transcription activation complex with two activators NtcA and NtcB
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.718 MDa / Experimental value: YES
Source (natural)Organism: Anabaena (bacteria) / Strain: PCC. 7120
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Details: 10 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl2 and 2 mM DTT
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMpotassium chlorideKCl1
25 mMmagnesium chlorideMgCl21
310 mM2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acidHEPES1
42 mMDithiothreitolDTT1
SpecimenConc.: 17 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Preliminary grid screening was performed manually.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5047
Details: Images were collected in movie-mode at 40 frames per second.

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Processing

EM software
IDNameCategory
1RELIONparticle selection
2EPUimage acquisition
4CTFFINDCTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
12RELIONclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 566246
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 65446 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT

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