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- PDB-8p2i: Cryo-EM structure of Pyrococcus furiosus apo form RNA polymerase ... -

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Basic information

Entry
Database: PDB / ID: 8p2i
TitleCryo-EM structure of Pyrococcus furiosus apo form RNA polymerase contracted clamp conformation with Spt4/5
Components
  • (DNA-directed RNA polymerase subunit ...) x 11
  • (Transcription elongation factor ...) x 2
KeywordsTRANSCRIPTION / RNA / Polymerase / Elongation / Complex / Pyrococcus furiosus / Archaea / DNA
Function / homology
Function and homology information


transcription elongation-coupled chromatin remodeling / RNA polymerase III activity / RNA polymerase II activity / tRNA transcription by RNA polymerase III / RNA polymerase I activity / translation elongation factor activity / DNA-directed RNA polymerase complex / regulation of DNA-templated transcription elongation / DNA-templated transcription initiation / ribonucleoside binding ...transcription elongation-coupled chromatin remodeling / RNA polymerase III activity / RNA polymerase II activity / tRNA transcription by RNA polymerase III / RNA polymerase I activity / translation elongation factor activity / DNA-directed RNA polymerase complex / regulation of DNA-templated transcription elongation / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / ribosome / structural constituent of ribosome / mRNA binding / nucleotide binding / DNA-templated transcription / regulation of DNA-templated transcription / regulation of transcription by RNA polymerase II / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm
Similarity search - Function
Archaeal transcription elongation factor Spt4 / Transcription elongation factor Spt5, archaeal / Archaeal transcription elongation factor Spt4 superfamily / DNA-directed RNA polymerase subunit Rpo4 / DNA-directed RNA polymerase subunit Rpo12 / DNA-directed RNA polymerase subunit Rpo1C / DNA-directed RNA polymerase subunit Rpo1N / DNA-directed RNA polymerase subunit Rpo2 / DNA-directed RNA polymerase subunit Rpo7 / Spt4/RpoE2 zinc finger ...Archaeal transcription elongation factor Spt4 / Transcription elongation factor Spt5, archaeal / Archaeal transcription elongation factor Spt4 superfamily / DNA-directed RNA polymerase subunit Rpo4 / DNA-directed RNA polymerase subunit Rpo12 / DNA-directed RNA polymerase subunit Rpo1C / DNA-directed RNA polymerase subunit Rpo1N / DNA-directed RNA polymerase subunit Rpo2 / DNA-directed RNA polymerase subunit Rpo7 / Spt4/RpoE2 zinc finger / Spt4/RpoE2 zinc finger / Spt4/RpoE2 zinc finger / HRDC domain superfamily / DNA-directed RNA polymerase, subunit E/RPC8 / NGN domain / Transcription elongation factor SPT5 / Early transcription elongation factor of RNA pol II, NGN section / NusG, N-terminal / In Spt5p, this domain may confer affinity for Spt4p. It possesses a RNP-like fold. / NusG, N-terminal domain superfamily / RNA polymerase subunit Rpb4/RPC9 / RNA polymerase Rpb4 / S1 domain profile. / HRDC-like superfamily / RNA polymerase Rpb7-like , N-terminal / RNA polymerase Rpb7-like, N-terminal domain superfamily / RNA polymerase subunit Rpb7-like / SHS2 domain found in N terminus of Rpb7p/Rpc25p/MJ0397 / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / : / DNA-directed RNA polymerase subunit RPABC5/Rpb10 / RNA polymerases, subunit N, zinc binding site / RNA polymerase subunit RPB10 / RNA polymerases N / 8 kDa subunit / RNA polymerases N / 8 Kd subunits signature. / DNA directed RNA polymerase, 7 kDa subunit / RNA polymerase archaeal subunit P/eukaryotic subunit RPABC4 / RNA polymerase, subunit H/Rpb5, conserved site / RNA polymerases H / 23 Kd subunits signature. / RNA polymerase subunit CX / DNA-directed RNA polymerase, 30-40kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo3/Rpb3/RPAC1 / RNA polymerases D / 30 to 40 Kd subunits signature. / DNA-directed RNA polymerase Rpb11, 13-16kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo11 / RNA polymerases L / 13 to 16 Kd subunits signature. / DNA-directed RNA polymerase subunit Rpo5/Rpb5 / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / DNA-directed RNA polymerase, RBP11-like dimerisation domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase, subunit H/Rpb5 C-terminal / RPB5-like RNA polymerase subunit superfamily / RNA polymerase Rpb5, C-terminal domain / Archaeal Rpo6/eukaryotic RPB6 RNA polymerase subunit / DNA-directed RNA polymerase, 14-18kDa subunit, conserved site / RNA polymerases K / 14 to 18 Kd subunits signature. / Ribosomal protein S1-like RNA-binding domain / S1 RNA binding domain / S1 domain / 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.
Similarity search - Domain/homology
DNA-directed RNA polymerase subunit Rpo1N / DNA-directed RNA polymerase subunit Rpo10 / DNA-directed RNA polymerase subunit Rpo12 / Transcription elongation factor Spt5 / DNA-directed RNA polymerase subunit Rpo3 / DNA-directed RNA polymerase subunit Rpo6 / DNA-directed RNA polymerase subunit Rpo5 / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit Rpo1C / DNA-directed RNA polymerase subunit Rpo4 ...DNA-directed RNA polymerase subunit Rpo1N / DNA-directed RNA polymerase subunit Rpo10 / DNA-directed RNA polymerase subunit Rpo12 / Transcription elongation factor Spt5 / DNA-directed RNA polymerase subunit Rpo3 / DNA-directed RNA polymerase subunit Rpo6 / DNA-directed RNA polymerase subunit Rpo5 / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit Rpo1C / DNA-directed RNA polymerase subunit Rpo4 / DNA-directed RNA polymerase subunit Rpo7 / Transcription elongation factor Spt4 / DNA-directed RNA polymerase subunit Rpo11
Similarity search - Component
Biological speciesPyrococcus furiosus DSM 3638 (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsTarau, D.M. / Reichelt, R. / Heiss, F.B. / Pilsl, M. / Hausner, W. / Engel, C. / Grohmann, D.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)SFB960 Germany
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Structural basis of archaeal RNA polymerase transcription elongation and Spt4/5 recruitment.
Authors: Daniela Tarău / Felix Grünberger / Michael Pilsl / Robert Reichelt / Florian Heiß / Sabine König / Henning Urlaub / Winfried Hausner / Christoph Engel / Dina Grohmann /
Abstract: Archaeal transcription is carried out by a multi-subunit RNA polymerase (RNAP) that is highly homologous in structure and function to eukaryotic RNAP II. Among the set of basal transcription factors, ...Archaeal transcription is carried out by a multi-subunit RNA polymerase (RNAP) that is highly homologous in structure and function to eukaryotic RNAP II. Among the set of basal transcription factors, only Spt5 is found in all domains of life, but Spt5 has been shaped during evolution, which is also reflected in the heterodimerization of Spt5 with Spt4 in Archaea and Eukaryotes. To unravel the mechanistic basis of Spt4/5 function in Archaea, we performed structure-function analyses using the archaeal transcriptional machinery of Pyrococcus furiosus (Pfu). We report single-particle cryo-electron microscopy reconstructions of apo RNAP and the archaeal elongation complex (EC) in the absence and presence of Spt4/5. Surprisingly, Pfu Spt4/5 also binds the RNAP in the absence of nucleic acids in a distinct super-contracted conformation. We show that the RNAP clamp/stalk module exhibits conformational flexibility in the apo state of RNAP and that the enzyme contracts upon EC formation or Spt4/5 engagement. We furthermore identified a contact of the Spt5-NGN domain with the DNA duplex that stabilizes the upstream boundary of the transcription bubble and impacts Spt4/5 activity in vitro. This study, therefore, provides the structural basis for Spt4/5 function in archaeal transcription and reveals a potential role beyond the well-described support of elongation.
History
DepositionMay 16, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 24, 2024Provider: repository / Type: Initial release
Revision 1.1May 15, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jun 19, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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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 Rpo1N
B: DNA-directed RNA polymerase subunit beta
C: DNA-directed RNA polymerase subunit Rpo1C
D: DNA-directed RNA polymerase subunit Rpo3
E: DNA-directed RNA polymerase subunit Rpo7
F: DNA-directed RNA polymerase subunit Rpo4
H: DNA-directed RNA polymerase subunit Rpo5
K: DNA-directed RNA polymerase subunit Rpo6
L: DNA-directed RNA polymerase subunit Rpo11
N: DNA-directed RNA polymerase subunit Rpo10
P: DNA-directed RNA polymerase subunit Rpo12
G: Transcription elongation factor Spt5
I: Transcription elongation factor Spt4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)407,21120
Polymers406,79413
Non-polymers4177
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area60070 Å2
ΔGint-342 kcal/mol
Surface area124130 Å2
MethodPISA

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Components

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DNA-directed RNA polymerase subunit ... , 11 types, 11 molecules ABCDEFHKLNP

#1: Protein DNA-directed RNA polymerase subunit Rpo1N / DNA-directed RNA polymerase subunit A'


Mass: 103612.250 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Gene: rpoA1, rpo1N, PFDSM3638_07875 / Production host: Pyrococcus furiosus DSM 3638 (archaea)
References: UniProt: A0A5C0XPL7, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta


Mass: 127188.898 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Gene: PF1564 / Production host: Pyrococcus furiosus DSM 3638 (archaea) / References: UniProt: Q8U0M3
#3: Protein DNA-directed RNA polymerase subunit Rpo1C / DNA-directed RNA polymerase subunit A''


Mass: 44468.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Gene: rpo1C, rpoA2, PF1562 / Production host: Pyrococcus furiosus DSM 3638 (archaea) / References: UniProt: Q8U0M5, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit Rpo3 / DNA-directed RNA polymerase subunit D


Mass: 31688.195 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Gene: rpo3, rpoD, PF1647 / Production host: Pyrococcus furiosus DSM 3638 (archaea) / References: UniProt: Q8U0E4, DNA-directed RNA polymerase
#5: Protein DNA-directed RNA polymerase subunit Rpo7


Mass: 21731.455 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Gene: rpo7 / Production host: Pyrococcus furiosus DSM 3638 (archaea) / References: UniProt: Q8U439
#6: Protein DNA-directed RNA polymerase subunit Rpo4 / DNA-directed RNA polymerase subunit F


Mass: 14116.311 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Gene: rpo4, rpoF, PF1036 / Production host: Pyrococcus furiosus DSM 3638 (archaea) / References: UniProt: Q8U216, DNA-directed RNA polymerase
#7: Protein DNA-directed RNA polymerase subunit Rpo5 / DNA-directed RNA polymerase subunit H


Mass: 9260.792 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Gene: rpo5, rpoH, PF1565 / Production host: Pyrococcus furiosus DSM 3638 (archaea) / References: UniProt: Q8U0M2, DNA-directed RNA polymerase
#8: Protein DNA-directed RNA polymerase subunit Rpo6 / DNA-directed RNA polymerase subunit K


Mass: 6243.490 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Gene: rpo6, rpoK, PF1642 / Production host: Pyrococcus furiosus DSM 3638 (archaea) / References: UniProt: Q8U0E8, DNA-directed RNA polymerase
#9: Protein DNA-directed RNA polymerase subunit Rpo11 / DNA-directed RNA polymerase subunit L


Mass: 11132.764 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Gene: rpo11, rpoL, PF0050 / Production host: Pyrococcus furiosus DSM 3638 (archaea) / References: UniProt: Q8U4N1, DNA-directed RNA polymerase
#10: Protein DNA-directed RNA polymerase subunit Rpo10 / DNA-directed RNA polymerase subunit N


Mass: 7800.158 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Gene: rpo10, rpoN, PF1643 / Production host: Pyrococcus furiosus DSM 3638 (archaea) / References: UniProt: P60292, DNA-directed RNA polymerase
#11: Protein/peptide DNA-directed RNA polymerase subunit Rpo12 / DNA-directed RNA polymerase subunit P


Mass: 5770.050 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Gene: rpo12, rpoP, PF2009 / Production host: Pyrococcus furiosus DSM 3638 (archaea) / References: UniProt: Q8TZI3, DNA-directed RNA polymerase

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Transcription elongation factor ... , 2 types, 2 molecules GI

#12: Protein Transcription elongation factor Spt5


Mass: 16816.600 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Gene: spt5, PF1990 / Production host: Pyrococcus furiosus DSM 3638 (archaea) / References: UniProt: Q8TZK1
#13: Protein Transcription elongation factor Spt4


Mass: 6965.045 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus DSM 3638 (archaea) / Gene: spt4, PF0255 / Production host: Pyrococcus furiosus DSM 3638 (archaea) / References: UniProt: Q8U440

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Non-polymers , 2 types, 7 molecules

#14: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#15: 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

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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: Pyrococcus furiosus apo form of RNA polymerase open clamp conformation
Type: COMPLEX / Entity ID: #1-#13 / Source: RECOMBINANT
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Source (recombinant)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 200
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 1 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 107970 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00426575
ELECTRON MICROSCOPYf_angle_d0.58235898
ELECTRON MICROSCOPYf_dihedral_angle_d4.5073620
ELECTRON MICROSCOPYf_chiral_restr0.0443991
ELECTRON MICROSCOPYf_plane_restr0.0054661

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