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- PDB-8e82: Mycobacterium tuberculosis RNAP elongation complex with NusG tran... -

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Basic information

Entry
Database: PDB / ID: 8.0E+82
TitleMycobacterium tuberculosis RNAP elongation complex with NusG transcription factor
Components
  • (DNA (54-MER)) x 2
  • (DNA-directed RNA polymerase subunit ...) x 4
  • RNA (5'-R(P*CP*GP*GP*AP*GP*AP*GP*GP*UP*A)-3')
  • Transcription termination/antitermination protein NusG
KeywordsTRANSCRIPTION/DNA/RNA / RNA polymerase / transcription factor / elongation / pausing / transcription-transferase-DNA complex / TRANSCRIPTION-DNA-RNA complex
Function / homology
Function and homology information


transcription elongation-coupled chromatin remodeling / DNA-directed RNA polymerase complex / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription termination / ribonucleoside binding / : / : / : / : ...transcription elongation-coupled chromatin remodeling / DNA-directed RNA polymerase complex / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription termination / ribonucleoside binding / : / : / : / : / : / : / DNA-directed RNA polymerase / protein dimerization activity / magnesium ion binding / DNA binding / zinc ion binding / cytosol / cytoplasm
Similarity search - Function
: / Transcription antitermination protein, NusG / Transcription antitermination protein, NusG, bacteria, conserved site / Transcription termination factor nusG signature. / NusG-like / Transcription termination factor nusG / Dna-directed Rna Polymerase Ii 140kd Polypeptide; Chain: B; domain 3 / RNA polymerase Rpb2, domain 2 / NusG, N-terminal / In Spt5p, this domain may confer affinity for Spt4p. It possesses a RNP-like fold. ...: / Transcription antitermination protein, NusG / Transcription antitermination protein, NusG, bacteria, conserved site / Transcription termination factor nusG signature. / NusG-like / Transcription termination factor nusG / Dna-directed Rna Polymerase Ii 140kd Polypeptide; Chain: B; domain 3 / RNA polymerase Rpb2, domain 2 / NusG, N-terminal / In Spt5p, this domain may confer affinity for Spt4p. It possesses a RNP-like fold. / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1 funnel domain / NusG, N-terminal domain superfamily / Enzyme I; Chain A, domain 2 / Topoisomerase I; Chain A, domain 4 / DNA-directed RNA polymerase, insert domain / RNA Polymerase Alpha Subunit; Chain A, domain 2 / 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 / 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 / Translation protein SH3-like domain superfamily / Ribosomal protein L2, domain 2 / Alpha-Beta Complex / Orthogonal Bundle / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / Transcription termination/antitermination protein NusG / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit alpha
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.03 Å
AuthorsDelbeau, M. / Darst, S.A. / Campbell, E.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Mol Cell / Year: 2023
Title: Structural and functional basis of the universal transcription factor NusG pro-pausing activity in Mycobacterium tuberculosis.
Authors: Madeleine Delbeau / Expery O Omollo / Ruby Froom / Steven Koh / Rachel A Mooney / Mirjana Lilic / Joshua J Brewer / Jeremy Rock / Seth A Darst / Elizabeth A Campbell / Robert Landick /
Abstract: Transcriptional pauses mediate regulation of RNA biogenesis. DNA-encoded pause signals trigger pausing by stabilizing RNA polymerase (RNAP) swiveling and inhibiting DNA translocation. The N-terminal ...Transcriptional pauses mediate regulation of RNA biogenesis. DNA-encoded pause signals trigger pausing by stabilizing RNA polymerase (RNAP) swiveling and inhibiting DNA translocation. The N-terminal domain (NGN) of the only universal transcription factor, NusG/Spt5, modulates pausing through contacts to RNAP and DNA. Pro-pausing NusGs enhance pauses, whereas anti-pausing NusGs suppress pauses. Little is known about pausing and NusG in the human pathogen Mycobacterium tuberculosis (Mtb). We report that MtbNusG is pro-pausing. MtbNusG captures paused, swiveled RNAP by contacts to the RNAP protrusion and nontemplate-DNA wedged between the NGN and RNAP gate loop. In contrast, anti-pausing Escherichia coli (Eco) NGN contacts the MtbRNAP gate loop, inhibiting swiveling and pausing. Using CRISPR-mediated genetics, we show that pro-pausing NGN is required for mycobacterial fitness. Our results define an essential function of mycobacterial NusG and the structural basis of pro- versus anti-pausing NusG activity, with broad implications for the function of all NusG orthologs.
History
DepositionAug 25, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 22, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 11, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Nov 13, 2024Group: Data collection / Structure summary
Category: 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

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
Z: Transcription termination/antitermination protein NusG
P: DNA (54-MER)
Q: DNA (54-MER)
R: RNA (5'-R(P*CP*GP*GP*AP*GP*AP*GP*GP*UP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)428,99512
Polymers428,8399
Non-polymers1553
Water00
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-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: 37745.328 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoA / Production host: Escherichia coli (E. coli) / References: UniProt: A5U8D3, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 129372.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: rpoB, MRA_0676 / Production host: Escherichia coli (E. coli) / References: UniProt: A5U052, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 147097.094 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria)
Gene: rpoC, E5M52_05480, ERS007665_00591, ERS013471_00574, ERS023446_00410, ERS075361_00813, ERS094182_01340, F6W99_03342
Production host: Escherichia coli (E. coli)
References: UniProt: A0A045J9E2, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11819.075 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria)
Gene: rpoZ, ERS007703_04032, ERS007720_04749, ERS094182_01030
Production host: Escherichia coli (E. coli)
References: UniProt: A0A0T9N9K3, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules PQ

#6: DNA chain DNA (54-MER)


Mass: 16521.557 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#7: DNA chain DNA (54-MER)


Mass: 16589.572 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / RNA chain , 2 types, 2 molecules ZR

#5: Protein Transcription termination/antitermination protein NusG


Mass: 25439.439 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria) / Gene: nusG / Production host: Escherichia coli (E. coli) / References: UniProt: A0A045HU92
#8: RNA chain RNA (5'-R(P*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)

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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
Has protein modificationY

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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 RNAP elongation complex with NusG transcription factor
Type: COMPLEX / Entity ID: #1-#8 / Source: MULTIPLE SOURCES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Mycobacterium tuberculosis (bacteria)1773
31synthetic construct (others)32630
Source (recombinant)Organism: Escherichia coli (E. coli)
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 / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 64.78 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.03 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 132324 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00325470
ELECTRON MICROSCOPYf_angle_d0.56134807
ELECTRON MICROSCOPYf_dihedral_angle_d14.1629764
ELECTRON MICROSCOPYf_chiral_restr0.1053976
ELECTRON MICROSCOPYf_plane_restr0.0044343

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