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- PDB-6j9f: Cryo-EM structure of Xanthomonos oryzae transcription elongation ... -

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Basic information

Entry
Database: PDB / ID: 6j9f
TitleCryo-EM structure of Xanthomonos oryzae transcription elongation complex with the bacteriophage protein P7
Components
  • (DNA (29-MER)) x 2
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • 45L
  • RNA (5'-R(P*AP*GP*CP*GP*GP*AP*GP*AP*GP*GP*UP*A)-3')
KeywordsTRANSCRIPTION / RNA polymerase / transcription termination / anti-termination / RNAP clamp / phage / transcription initiation / P7 / Xanthomonos oryzae / Xp10
Function / homology
Function and homology information


DNA-directed RNA polymerase complex / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm
Similarity search - Function
Double Stranded RNA Binding Domain - #560 / : / Xanthomonas phage Xp10 Transcription regulator P7 / : / RNA polymerase subunit, RPB6/omega / Eukaryotic RPB6 RNA polymerase subunit / DNA-directed RNA polymerase, insert domain / RNA polymerase, RBP11-like subunit / RNA Polymerase Alpha Subunit; Chain A, domain 2 / Gyrase A; domain 2 ...Double Stranded RNA Binding Domain - #560 / : / Xanthomonas phage Xp10 Transcription regulator P7 / : / RNA polymerase subunit, RPB6/omega / Eukaryotic RPB6 RNA polymerase subunit / DNA-directed RNA polymerase, insert domain / RNA polymerase, RBP11-like subunit / RNA Polymerase Alpha Subunit; Chain A, domain 2 / Gyrase A; domain 2 / Double Stranded RNA Binding Domain / 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 / Beta Complex / 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 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 / Alpha-Beta Complex / 2-Layer Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit omega / RNA polymerase inhibitor p7
Similarity search - Component
Biological speciesXanthomonas oryzae pv. oryzae PXO99A (bacteria)
Xanthomonas oryzae pv. oryzae MAFF 311018 (bacteria)
Xanthomonas oryzae pv. oryzae (bacteria)
Xanthomonas virus Xp10
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.95 Å
AuthorsYou, L.L. / Zhang, Y.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China31822001 China
CitationJournal: Nat Commun / Year: 2019
Title: Structural basis for transcription antitermination at bacterial intrinsic terminator.
Authors: Linlin You / Jing Shi / Liqiang Shen / Lingting Li / Chengli Fang / Chengzhi Yu / Wenbo Cheng / Yu Feng / Yu Zhang /
Abstract: Bacteriophages typically hijack the host bacterial transcriptional machinery to regulate their own gene expression and that of the host bacteria. The structural basis for bacteriophage protein- ...Bacteriophages typically hijack the host bacterial transcriptional machinery to regulate their own gene expression and that of the host bacteria. The structural basis for bacteriophage protein-mediated transcription regulation-in particular transcription antitermination-is largely unknown. Here we report the 3.4 Å and 4.0 Å cryo-EM structures of two bacterial transcription elongation complexes (P7-NusA-TEC and P7-TEC) comprising the bacteriophage protein P7, a master host-transcription regulator encoded by bacteriophage Xp10 of the rice pathogen Xanthomonas oryzae pv. Oryzae (Xoo) and discuss the mechanisms by which P7 modulates the host bacterial RNAP. The structures together with biochemical evidence demonstrate that P7 prevents transcription termination by plugging up the RNAP RNA-exit channel and impeding RNA-hairpin formation at the intrinsic terminator. Moreover, P7 inhibits transcription initiation by restraining RNAP-clamp motions. Our study reveals the structural basis for transcription antitermination by phage proteins and provides insights into bacterial transcription regulation.
History
DepositionJan 22, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 17, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 31, 2019Group: Data collection / 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_PubMed / _citation.title / _citation_author.name
Revision 1.2Nov 6, 2019Group: Data collection / Other / Category: cell / Item: _cell.Z_PDB
Revision 1.3Mar 27, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-9786
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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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
G: DNA (29-MER)
H: DNA (29-MER)
I: RNA (5'-R(P*AP*GP*CP*GP*GP*AP*GP*AP*GP*GP*UP*A)-3')
J: 45L
hetero molecules


Theoretical massNumber of molelcules
Total (without water)429,92812
Polymers429,7739
Non-polymers1553
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area38810 Å2
ΔGint-237 kcal/mol
Surface area142880 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 / Polymerase / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 38030.168 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xanthomonas oryzae pv. oryzae PXO99A (bacteria)
Gene: rpoA / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: B2SQT4, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / Polymerase / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 154471.719 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xanthomonas oryzae pv. oryzae MAFF 311018 (bacteria)
Strain: MAFF 311018 / Gene: rpoB, XOO3394 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: Q2NZX8, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 155444.609 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xanthomonas oryzae pv. oryzae PXO99A (bacteria)
Gene: rpoC / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: B2SQQ2, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11112.536 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xanthomonas oryzae pv. oryzae (bacteria)
Gene: rpoZ / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
References: UniProt: A0A0U4VN94, UniProt: Q5H3R9*PLUS, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules GH

#5: DNA chain DNA (29-MER)


Mass: 8813.646 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: DNA chain DNA (29-MER)


Mass: 8840.689 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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RNA chain / Protein , 2 types, 2 molecules IJ

#7: RNA chain RNA (5'-R(P*AP*GP*CP*GP*GP*AP*GP*AP*GP*GP*UP*A)-3')


Mass: 6509.968 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#8: Protein 45L / RNA polymerase inhibitor p7


Mass: 8519.374 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xanthomonas virus Xp10 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: Q8LTJ5

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Non-polymers , 2 types, 3 molecules

#9: Chemical ChemComp-MG / MAGNESIUM ION


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


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

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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: Xoo transcription elongation complex with P7(P7-TEC) / Type: COMPLEX / Entity ID: #1-#8 / Source: RECOMBINANT
Molecular weightValue: 0.427 MDa
Source (natural)Organism: Xanthomonas oryzae (bacteria) / Strain: PXO99A
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHepesHepes1
250 mMPotassium ChlorideKCL1
35 mMMagnesium chlorideMgCL21
43 mMDL-DithiothreitolDTT1
SpecimenConc.: 9.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3 4C
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283 K / Details: blot for 8 seconds before plunging

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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 FIELDBright-field microscopy / Nominal defocus max: 2600 nm / Nominal defocus min: 2000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 8 sec. / Electron dose: 1.675 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2271
Image scansMovie frames/image: 32

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Processing

EM software
IDNameVersionCategoryDetailsImage processing-ID
2SerialEMimage acquisition
4CTFFIND4CTF correctionCTFFIND4 was used to Contrast-transfer-function estimations1
7UCSF Chimera1.10.2model fitting
9PHENIX1.14model refinement
10RELION3initial Euler assignment1
11RELION3final Euler assignment1
12RELION3classification1
13RELION33D reconstruction1
14CTFFIND4particle selection2
21CTFFIND4CTF correctionCTFFIND4 was used to Contrast-transfer-function estimations2
23RELION3initial Euler assignment2
24RELION3final Euler assignment2
25RELION3classification2
26RELION33D reconstruction2
Image processing
IDImage recording-ID
11
21
CTF correction
IDEM image processing-IDType
11NONE
22NONE
Particle selection
IDImage processing-IDNum. of particles selected
11410547
22410547
Symmetry
IDImage processing-IDEntry-IDPoint symmetry
116J9FC1 (asymmetric)
226J9FC1 (asymmetric)
3D reconstruction

Algorithm: FOURIER SPACE / Entry-ID: 6J9F / Num. of particles: 319613 / Resolution: 3.95 Å / Resolution method: FSC 0.143 CUT-OFF / Symmetry type: POINT

IDImage processing-IDNum. of class averages
111
22
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
15UPC

5upc
PDB Unreleased entry

15UPC1PDBexperimental model
22MC6

2mc6

12MC62PDBexperimental model

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