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- PDB-8eos: M. tuberculosis RNAP elongation complex with NusG and CMPCPP -

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Basic information

Entry
Database: PDB / ID: 8eos
TitleM. tuberculosis RNAP elongation complex with NusG and CMPCPP
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • DNA (36-MER)
  • DNA (38-MER)
  • RNA (5'-R(P*AP*AP*GP*CP*GP*GP*AP*GP*AP*GP*GP*UP*A)-3')
  • Transcription termination/antitermination protein NusG
KeywordsTRANSCRIPTION / Transcription elongation RNA polymerase pausing NusG cryo-EM
Function / homology
Function and homology information


Antimicrobial action and antimicrobial resistance in Mtb / transcription elongation-coupled chromatin remodeling / DNA-directed RNA polymerase complex / peptidoglycan-based cell wall / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription termination / ribonucleoside binding / : / : ...Antimicrobial action and antimicrobial resistance in Mtb / transcription elongation-coupled chromatin remodeling / DNA-directed RNA polymerase complex / peptidoglycan-based cell wall / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription termination / ribonucleoside binding / : / : / : / : / : / : / DNA-directed RNA polymerase / protein dimerization activity / response to antibiotic / magnesium ion binding / DNA binding / zinc ion binding / plasma membrane / 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 / 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 ...: / Transcription antitermination protein, NusG / Transcription antitermination protein, NusG, bacteria, conserved site / Transcription termination factor nusG signature. / NusG-like / Transcription termination factor nusG / 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 / Orthogonal Bundle / Mainly Beta / Mainly Alpha
Similarity search - Domain/homology
Chem-2TM / DNA / DNA (> 10) / RNA / RNA (> 10) / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit alpha / Transcription termination/antitermination protein NusG
Similarity search - Component
Biological speciesMycobacterium tuberculosis H37Rv (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsVishwakarma, R.K. / Murakami, K.S.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131860 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM098399 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Allosteric mechanism of transcription inhibition by NusG-dependent pausing of RNA polymerase.
Authors: Rishi K Vishwakarma / M Zuhaib Qayyum / Paul Babitzke / Katsuhiko S Murakami /
Abstract: NusG is a transcription elongation factor that stimulates transcription pausing in Gram+ bacteria including by sequence-specific interaction with a conserved pause-inducing TTNTTT motif found in the ...NusG is a transcription elongation factor that stimulates transcription pausing in Gram+ bacteria including by sequence-specific interaction with a conserved pause-inducing TTNTTT motif found in the non-template DNA (ntDNA) strand within the transcription bubble. To reveal the structural basis of NusG-dependent pausing, we determined a cryo-EM structure of a paused transcription complex (PTC) containing RNA polymerase (RNAP), NusG, and the TTNTTT motif in the ntDNA strand. The interaction of NusG with the ntDNA strand rearranges the transcription bubble by positioning three consecutive T residues in a cleft between NusG and the β-lobe domain of RNAP. We revealed that the RNAP swivel module rotation (swiveling), which widens (swiveled state) and narrows (non-swiveled state) a cleft between NusG and the β-lobe, is an intrinsic motion of RNAP and is directly linked to trigger loop (TL) folding, an essential conformational change of all cellular RNAPs for the RNA synthesis reaction. We also determined cryo-EM structures of RNAP escaping from the paused transcription state. These structures revealed the NusG-dependent pausing mechanism by which NusG-ntDNA interaction inhibits the transition from swiveled to non-swiveled states, thereby preventing TL folding and RNA synthesis allosterically. This motion is also reduced by the formation of an RNA hairpin within the RNA exit channel. Thus, the pause half-life can be modulated by the strength of the NusG-ntDNA interaction and/or the stability of the RNA hairpin. NusG residues that interact with the TTNTTT motif are widely conserved in bacteria, suggesting that NusG-dependent pausing is widespread.
History
DepositionOct 4, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 1, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 22, 2023Group: Database references / Category: 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 / _citation_author.identifier_ORCID
Revision 1.2Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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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
G: Transcription termination/antitermination protein NusG
T: DNA (38-MER)
N: DNA (36-MER)
R: RNA (5'-R(P*AP*AP*GP*CP*GP*GP*AP*GP*AP*GP*GP*UP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)421,50814
Polymers420,8489
Non-polymers6615
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
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 H37Rv (bacteria)
Strain: ATCC 25618 / H37Rv / Gene: rpoA, Rv3457c, MTCY13E12.10c / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P9WGZ1, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 130018.828 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Strain: ATCC 25618 / H37Rv / Gene: rpoB, Rv0667, MTCI376.08c / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P9WGY9, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 146968.969 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Strain: ATCC 25618 / H37Rv / Gene: rpoC, Rv0668, MTCI376.07c / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P9WGY7, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11851.140 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Strain: ATCC 25618 / H37Rv / Gene: rpoZ, Rv1390, MTCY21B4.07 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P9WGY5, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules TN

#6: DNA chain DNA (38-MER)


Mass: 12211.804 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#7: DNA chain DNA (36-MER)


Mass: 12324.892 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 GR

#5: Protein Transcription termination/antitermination protein NusG


Mass: 25471.504 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Strain: ATCC 25618 / H37Rv / Gene: nusG, Rv0639, MTCY20H10.20 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P9WIU9
#8: RNA chain RNA (5'-R(P*AP*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)

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Non-polymers , 3 types, 5 molecules

#9: Chemical ChemComp-2TM / 5'-O-[(S)-hydroxy{[(S)-hydroxy(phosphonooxy)phosphoryl]methyl}phosphoryl]cytidine / CMPcPP


Mass: 481.184 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H18N3O13P3
#10: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / 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: Transcription elongation complex with M. tuberculosis NusG and CMPCPP
Type: COMPLEX
Details: It contains M. tuberculosis RNA polymerase, DNA/RNA scaffold, M. tuberculosis NusG and CMPCPP
Entity ID: #1-#8 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.9
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K
Details: Blot for 4-5 seconds before plunging in liquid 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: OTHER
Electron lensMode: OTHER / Nominal defocus max: 2500 nm / Nominal defocus min: 750 nm
Image recordingElectron dose: 45 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM softwareName: cryoSPARC / Category: CTF correction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 129716 / Symmetry type: POINT
RefinementCross valid method: NONE
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004326226
ELECTRON MICROSCOPYf_angle_d0.509435922
ELECTRON MICROSCOPYf_chiral_restr0.04164098
ELECTRON MICROSCOPYf_plane_restr0.00414418
ELECTRON MICROSCOPYf_dihedral_angle_d14.846210062

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