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- PDB-7d7d: CryoEM structure of gp45-dependent transcription activation complex -

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Basic information

Entry
Database: PDB / ID: 7d7d
TitleCryoEM structure of gp45-dependent transcription activation complex
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • DNA (nontemplate strand)
  • DNA (template strand)
  • DNA polymerase clamp
  • RNA polymerase-associated protein Gp33
  • gp55
KeywordsTRANSCRIPTION / RNA polymerase
Function / homology
Function and homology information


late viral transcription / bidirectional double-stranded viral DNA replication / transcription regulator activator activity / viral DNA genome replication / DNA polymerase processivity factor activity / viral transcription / DNA-directed RNA polymerase activity / DNA-directed RNA polymerase complex / ribonucleoside binding / DNA-directed RNA polymerase ...late viral transcription / bidirectional double-stranded viral DNA replication / transcription regulator activator activity / viral DNA genome replication / DNA polymerase processivity factor activity / viral transcription / DNA-directed RNA polymerase activity / DNA-directed RNA polymerase complex / ribonucleoside binding / DNA-directed RNA polymerase / DNA replication / protein dimerization activity / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm
Similarity search - Function
Late transcription coactivator / Phage late-transcription coactivator superfamily / Late transcription coactivator, myovirus / Phage late-transcription coactivator / Sliding clamp, C-terminal / Sliding clamp / gp45 sliding clamp, C terminal / DNA polymerase processivity factor / DNA polymerase processivity factor / : ...Late transcription coactivator / Phage late-transcription coactivator superfamily / Late transcription coactivator, myovirus / Phage late-transcription coactivator / Sliding clamp, C-terminal / Sliding clamp / gp45 sliding clamp, C terminal / DNA polymerase processivity factor / DNA polymerase processivity factor / : / : / 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, 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
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit beta' / Sliding clamp / Late transcription coactivator / DNA-directed RNA polymerase subunit alpha
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Escherichia coli 1-392-07_S4_C3 (bacteria)
Escherichia virus T4
Enterobacteria phage T4 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsShi, J. / Wen, A. / Jin, S. / Feng, Y.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31970040 China
CitationJournal: Nat Commun / Year: 2021
Title: Transcription activation by a sliding clamp.
Authors: Jing Shi / Aijia Wen / Sha Jin / Bo Gao / Yang Huang / Yu Feng /
Abstract: Transcription activation of bacteriophage T4 late genes is accomplished by a transcription activation complex containing RNA polymerase (RNAP), the promoter specificity factor gp55, the coactivator ...Transcription activation of bacteriophage T4 late genes is accomplished by a transcription activation complex containing RNA polymerase (RNAP), the promoter specificity factor gp55, the coactivator gp33, and a universal component of cellular DNA replication, the sliding clamp gp45. Although genetic and biochemical studies have elucidated many aspects of T4 late gene transcription, no precise structure of the transcription machinery in the process is available. Here, we report the cryo-EM structures of a gp55-dependent RNAP-promoter open complex and an intact gp45-dependent transcription activation complex. The structures reveal the interactions between gp55 and the promoter DNA that mediate the recognition of T4 late promoters. In addition to the σR2 homology domain, gp55 has a helix-loop-helix motif that chaperons the template-strand single-stranded DNA of the transcription bubble. Gp33 contacts both RNAP and the upstream double-stranded DNA. Gp45 encircles the DNA and tethers RNAP to it, supporting the idea that gp45 switches the promoter search from three-dimensional diffusion mode to one-dimensional scanning mode.
History
DepositionOct 3, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 20, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 17, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID
Revision 1.2Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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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'
T: DNA (template strand)
F: gp55
N: DNA (nontemplate strand)
K: RNA polymerase-associated protein Gp33
E: DNA-directed RNA polymerase subunit omega
G: DNA polymerase clamp
H: DNA polymerase clamp
I: DNA polymerase clamp
hetero molecules


Theoretical massNumber of molelcules
Total (without water)540,50615
Polymers540,35112
Non-polymers1553
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area47150 Å2
ΔGint-232 kcal/mol
Surface area194110 Å2

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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: 36558.680 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) / References: UniProt: U9ZUN7, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 150820.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli 1-392-07_S4_C3 (bacteria)
Gene: rpoB, AD40_4833 / Production host: Escherichia coli (E. coli)
References: UniProt: A0A080FHH4, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 155366.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) / References: UniProt: D7Y6A2, DNA-directed RNA polymerase
#8: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 10249.547 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli)
References: UniProt: A0A070UPX4, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules TN

#4: DNA chain DNA (template strand)


Mass: 18203.668 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia virus T4 / Production host: Escherichia virus T4
#6: DNA chain DNA (nontemplate strand)


Mass: 18155.713 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia virus T4

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Protein , 3 types, 5 molecules FKGHI

#5: Protein gp55


Mass: 21565.160 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia virus T4 / Production host: Escherichia coli (E. coli)
#7: Protein RNA polymerase-associated protein Gp33


Mass: 15014.942 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterobacteria phage T4 (virus) / Gene: 33 / Production host: Escherichia coli (E. coli) / References: UniProt: P13338
#9: Protein DNA polymerase clamp / DNA polymerase accessory protein 45 / DNA polymerase sliding clamp / Gp45


Mass: 25952.377 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterobacteria phage T4 (virus) / Gene: 45 / Production host: Escherichia coli (E. coli) / References: UniProt: P04525

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Non-polymers , 2 types, 3 molecules

#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#11: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN

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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: gp45-dependent transcription activation complex / Type: COMPLEX / Entity ID: #1-#9 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Thermus thermophilus (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
Image recordingElectron dose: 59 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 8981 / Symmetry type: POINT

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