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- PDB-8urw: Cyanobacterial RNA polymerase elongation complex with NusG and CTP -

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Basic information

Entry
Database: PDB / ID: 8urw
TitleCyanobacterial RNA polymerase elongation complex with NusG and CTP
Components
  • (DNA (40-MER)) x 2
  • (DNA-directed RNA polymerase subunit ...) x 5
  • RNA (5'-R(P*AP*AP*AP*GP*AP*GP*GP*AP*GP*AP*GP*GP*UP*A)-3')
  • Transcription termination/antitermination protein NusG
KeywordsTRANSCRIPTION / Transcription regulation / RNAP / NusG / cryo-EM
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 / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / DNA-templated transcription ...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 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
DNA-directed RNA polymerase, subunit gamma / DNA-directed RNA polymerase subunit RpoC1 / DNA-directed RNA polymerase, subunit beta'' / : / 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 ...DNA-directed RNA polymerase, subunit gamma / DNA-directed RNA polymerase subunit RpoC1 / DNA-directed RNA polymerase, subunit beta'' / : / 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, 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 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / 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, 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 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / 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 / KOW (Kyprides, Ouzounis, Woese) motif. / Translation protein SH3-like domain superfamily / KOW motif / KOW / Ribosomal protein L2, domain 2
Similarity search - Domain/homology
CYTIDINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / RNA / RNA (> 10) / DNA-directed RNA polymerase subunit gamma / 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 speciesSynechococcus elongatus (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.79 Å
AuthorsQayyum, M.Z. / Imashimizu, M. / Leanca, M. / Vishwakarma, R.K. / Bradley Riaz, A. / Yuzenkova, Y. / Murakami, K.S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131860 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Structure and function of the Si3 insertion integrated into the trigger loop/helix of cyanobacterial RNA polymerase.
Authors: M Zuhaib Qayyum / Masahiko Imashimizu / Miron Leanca / Rishi K Vishwakarma / Amber Riaz-Bradley / Yulia Yuzenkova / Katsuhiko S Murakami /
Abstract: Cyanobacteria and evolutionarily related chloroplasts of algae and plants possess unique RNA polymerases (RNAPs) with characteristics that distinguish them from canonical bacterial RNAPs. The largest ...Cyanobacteria and evolutionarily related chloroplasts of algae and plants possess unique RNA polymerases (RNAPs) with characteristics that distinguish them from canonical bacterial RNAPs. The largest subunit of cyanobacterial RNAP (cyRNAP) is divided into two polypeptides, β'1 and β'2, and contains the largest known lineage-specific insertion domain, Si3, located in the middle of the trigger loop and spanning approximately half of the β'2 subunit. In this study, we present the X-ray crystal structure of Si3 and the cryo-EM structures of the cyRNAP transcription elongation complex plus the NusG factor with and without incoming nucleoside triphosphate (iNTP) bound at the active site. Si3 has a well-ordered and elongated shape that exceeds the length of the main body of cyRNAP, fits into cavities of cyRNAP in the absence of iNTP bound at the active site and shields the binding site of secondary channel-binding proteins such as Gre and DksA. A small transition from the trigger loop to the trigger helix upon iNTP binding results in a large swing motion of Si3; however, this transition does not affect the catalytic activity of cyRNAP due to its minimal contact with cyRNAP, NusG, or DNA. This study provides a structural framework for understanding the evolutionary significance of these features unique to cyRNAP and chloroplast RNAP and may provide insights into the molecular mechanism of transcription in specific environment of photosynthetic organisms and organelle.
History
DepositionOct 26, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 27, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 21, 2024Group: 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 / _citation_author.name

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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 gamma
E: DNA-directed RNA polymerase subunit omega
Z: DNA-directed RNA polymerase subunit beta'
G: Transcription termination/antitermination protein NusG
N: DNA (40-MER)
T: DNA (40-MER)
R: RNA (5'-R(P*AP*AP*AP*GP*AP*GP*GP*AP*GP*AP*GP*GP*UP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)470,15814
Polymers469,52010
Non-polymers6384
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA-directed RNA polymerase subunit ... , 5 types, 6 molecules ABCDEZ

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


Mass: 33814.891 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechococcus elongatus (bacteria) / Gene: rpoA, Synpcc7942_2209 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q31L30, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 123428.547 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechococcus elongatus (bacteria) / Gene: rpoB, Synpcc7942_1522 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q31N17, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit gamma / RNAP subunit gamma / RNA polymerase subunit gamma / Transcriptase subunit gamma


Mass: 71054.367 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechococcus elongatus (bacteria) / Gene: rpoC1, Synpcc7942_1523 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P42079, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 8787.966 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechococcus elongatus (bacteria) / Gene: rpoZ, Synpcc7942_1710 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q31MH9, DNA-directed RNA polymerase
#5: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 144006.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechococcus elongatus (bacteria) / Gene: rpoC2, Synpcc7942_1524 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q31N15, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules NT

#7: DNA chain DNA (40-MER)


Mass: 12380.916 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#8: DNA chain DNA (40-MER)


Mass: 12251.828 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

#6: Protein Transcription termination/antitermination protein NusG


Mass: 23117.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechococcus elongatus (bacteria) / Gene: nusG, Synpcc7942_0635 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q31QK2
#9: RNA chain RNA (5'-R(P*AP*AP*AP*GP*AP*GP*GP*AP*GP*AP*GP*GP*UP*A)-3')


Mass: 6863.198 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 4 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
#12: Chemical ChemComp-CTP / CYTIDINE-5'-TRIPHOSPHATE


Mass: 483.156 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H16N3O14P3

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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: Cyanobacterial RNA polymerase elongation complex with NusG and CTP
Type: COMPLEX / Entity ID: #1-#9 / Source: RECOMBINANT
Source (natural)Organism: Synechococcus elongatus (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8 / Details: 40mM Tris-HCl 200mM KCl 1mM EDTA 1mM DTT 5mM MgCl2
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: OTHER / Nominal defocus max: 1750 nm / Nominal defocus min: 750 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 2.79 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 132172 / Symmetry type: POINT
RefinementCross valid method: NONE

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