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- PDB-9gur: 30S mRNA delivery complex TEC resolved (TEC only) -

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Basic information

Entry
Database: PDB / ID: 9gur
Title30S mRNA delivery complex TEC resolved (TEC only)
Components
  • (DNA-directed RNA polymerase subunit ...) x 5
  • Non-Template DNA strand
  • Template DNA strand
  • Transcription termination/antitermination protein NusG
  • mRNA
KeywordsRIBOSOME / Transcription / translation / coupling
Function / homology
Function and homology information


RNA polymerase complex / transcription elongation-coupled chromatin remodeling / submerged biofilm formation / cellular response to cell envelope stress / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / bacterial-type flagellum-dependent cell motility / nitrate assimilation ...RNA polymerase complex / transcription elongation-coupled chromatin remodeling / submerged biofilm formation / cellular response to cell envelope stress / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / bacterial-type flagellum-dependent cell motility / nitrate assimilation / transcription elongation factor complex / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription initiation / cell motility / DNA-templated transcription termination / ribonucleoside binding / : / : / : / : / : / : / DNA-directed RNA polymerase / ribosome biogenesis / response to heat / protein-containing complex assembly / intracellular iron ion homeostasis / protein dimerization activity / response to antibiotic / magnesium ion binding / DNA binding / zinc ion binding / 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. / NusG, N-terminal domain superfamily / DNA-directed RNA polymerase, omega subunit ...: / 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. / NusG, N-terminal domain superfamily / 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 / 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 Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, RBP11-like subunit / 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 / KOW (Kyprides, Ouzounis, Woese) motif. / Translation protein SH3-like domain superfamily / KOW motif / KOW / Ribosomal protein L2, domain 2
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / Transcription termination/antitermination protein NusG
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsRahil, H. / Weixlbaumer, A. / Webster, M.W.
Funding supportEuropean Union, France, United States, United Kingdom, 6items
OrganizationGrant numberCountry
European Research Council (ERC)679734European Union
Agence Nationale de la Recherche (ANR)ANR-10-LABX-0030-INRT France
Agence Nationale de la Recherche (ANR)ANR-10-IDEX-0002-02 France
European Molecular Biology Organization (EMBO)European Union
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM131922 United States
Wellcome Trust203149 United Kingdom
CitationJournal: Science / Year: 2024
Title: Molecular basis of mRNA delivery to the bacterial ribosome.
Authors: Michael W Webster / Adrien Chauvier / Huma Rahil / Andrea Graziadei / Kristine Charles / Nataliya Miropolskaya / Maria Takacs / Charlotte Saint-André / Juri Rappsilber / Nils G Walter / Albert Weixlbaumer /
Abstract: Protein synthesis begins with the formation of a ribosome-messenger RNA (mRNA) complex. In bacteria, the small ribosomal subunit (30) is recruited to many mRNAs through base pairing with the Shine- ...Protein synthesis begins with the formation of a ribosome-messenger RNA (mRNA) complex. In bacteria, the small ribosomal subunit (30) is recruited to many mRNAs through base pairing with the Shine-Dalgarno (SD) sequence and RNA binding by ribosomal protein bS1. Translation can initiate on nascent mRNAs, and RNA polymerase (RNAP) can promote the recruitment of the pioneering 30. Here, we examined 30 recruitment to nascent mRNAs using cryo-electron microscopy, single-molecule fluorescence colocalization, and in-cell cross-linking mass spectrometry. We show that bS1 delivers the mRNA to the ribosome for SD duplex formation and 30 activation. Additionally, bS1 and RNAP stimulate translation initiation. Our work provides a mechanistic framework for how the SD duplex, ribosomal proteins, and RNAP cooperate in 30 recruitment to mRNAs and establish transcription-translation coupling.
History
DepositionSep 20, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 20, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 11, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
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.identifier_ORCID / _citation_author.name / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
1: DNA-directed RNA polymerase subunit alpha
5: DNA-directed RNA polymerase subunit omega
6: Non-Template DNA strand
7: Template DNA strand
X: mRNA
2: DNA-directed RNA polymerase subunit alpha
3: DNA-directed RNA polymerase subunit beta
4: DNA-directed RNA polymerase subunit beta'
Z: Transcription termination/antitermination protein NusG
hetero molecules


Theoretical massNumber of molelcules
Total (without water)396,94512
Polymers396,7909
Non-polymers1553
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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DNA-directed RNA polymerase subunit ... , 5 types, 5 molecules 15234

#1: Protein DNA-directed RNA polymerase subunit alpha / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 25339.830 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: rpoA, pez, phs, sez, b3295, JW3257 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7Z4, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 8093.104 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: rpoZ, b3649, JW3624 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A800, DNA-directed RNA polymerase
#6: Protein DNA-directed RNA polymerase subunit alpha / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 25412.881 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: rpoA, pez, phs, sez, b3295, JW3257 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7Z4, DNA-directed RNA polymerase
#7: 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 K-12 (bacteria)
Gene: rpoB, groN, nitB, rif, ron, stl, stv, tabD, b3987, JW3950
Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A8V2, DNA-directed RNA polymerase
#8: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 150565.453 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: rpoC, tabB, b3988, JW3951 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A8T7, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules 67

#3: DNA chain Non-Template DNA strand


Mass: 9111.840 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain Template DNA strand


Mass: 9262.977 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 XZ

#5: RNA chain mRNA


Mass: 5158.191 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#9: Protein Transcription termination/antitermination protein NusG


Mass: 13025.049 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: nusG, b3982, JW3945 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0AFG0

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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
Has protein modificationN

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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 / Type: COMPLEX / Entity ID: #1-#9 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli K-12 (bacteria)
Buffer solutionpH: 7.4
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 49.95 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 7285 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00327761
ELECTRON MICROSCOPYf_angle_d0.55637830
ELECTRON MICROSCOPYf_dihedral_angle_d15.25310878
ELECTRON MICROSCOPYf_chiral_restr0.0764320
ELECTRON MICROSCOPYf_plane_restr0.0044692

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