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- PDB-6kf3: Cryo-EM structure of Thermococcus kodakarensis RNA polymerase -

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Basic information

Entry
Database: PDB / ID: 6kf3
TitleCryo-EM structure of Thermococcus kodakarensis RNA polymerase
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 8
  • (DNA-directed RNA polymerase, subunit ...Polymerase) x 2
  • DNA-directed RNA polymerase subunitPolymerase
KeywordsTRANSCRIPTION / apo-RNA polymerase
Function / homology
Function and homology information


RNA polymerase complex / DNA-templated transcription, initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / transcription, DNA-templated / protein dimerization activity / nucleotide binding / DNA binding / zinc ion binding / metal ion binding
Similarity search - Function
Alpha-Beta Plaits - #3110 / Helix Hairpins - #10 / 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 Rpo3 / DNA-directed RNA polymerase subunit Rpo4 / DNA-directed RNA polymerase subunit Rpo11 / HRDC domain ...Alpha-Beta Plaits - #3110 / Helix Hairpins - #10 / 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 Rpo3 / DNA-directed RNA polymerase subunit Rpo4 / DNA-directed RNA polymerase subunit Rpo11 / HRDC domain / DNA-directed RNA polymerase, subunit E/RPC8 / RNA polymerase Rpb7-like, N-terminal domain / RNA polymerase ii, chain L / RPB5-like RNA polymerase subunit / RNA polymerase subunit, RPB6/omega / Eukaryotic RPB6 RNA polymerase subunit / DNA-directed RNA polymerase, insert domain / RNA polymerase, RBP11-like subunit / RNA Polymerase Alpha Subunit; Chain A, domain 2 / RNA polymerase subunit Rpb4/RPC9 / RNA polymerase Rpb4 / Rubrerythrin, domain 2 / S1 domain profile. / RNA polymerase Rpb7-like , N-terminal / SHS2 domain found in N terminus of Rpb7p/Rpc25p/MJ0397 / HRDC-like superfamily / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb7-like, N-terminal domain superfamily / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 5 / RNA polymerase subunit RPB10 / RNA polymerases, subunit N, zinc binding site / RNA polymerases N / 8 Kd subunits signature. / DNA-directed RNA polymerase subunit RPABC5/Rpb10 / RNA polymerases N / 8 kDa subunit / Helix Hairpins / DNA directed RNA polymerase, 7 kDa subunit / RNA polymerase archaeal subunit P/eukaryotic subunit RPABC4 / RNA polymerase subunit CX / RNA polymerases D / 30 to 40 Kd subunits signature. / DNA-directed RNA polymerase, 30-40kDa subunit, conserved site / DNA-directed RNA polymerase, RBP11-like dimerisation domain / RNA polymerase Rpb5, C-terminal domain / RPB5-like RNA polymerase subunit superfamily / RNA polymerase, subunit H/Rpb5 C-terminal / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases L / 13 to 16 Kd subunits signature. / RNA polymerases H / 23 Kd subunits signature. / RNA polymerase, subunit H/Rpb5, conserved site / : / DNA-directed RNA polymerase Rpb11, 13-16kDa subunit, conserved site / : / DNA-directed RNA polymerase, 14-18kDa subunit, conserved site / RNA polymerases K / 14 to 18 Kd subunits signature. / Archaeal Rpo6/eukaryotic RPB6 RNA polymerase subunit / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / Gyrase A; domain 2 / S1 RNA binding domain / Ribosomal protein S1-like RNA-binding domain / RNA-binding domain, S1 / S1 domain / Homeodomain-like / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb6 / RPB6/omega subunit-like superfamily / RNA polymerase, subunit omega/Rpo6/RPB6 / Single Sheet / RNA polymerase Rpb2, domain 2 superfamily / : / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb1, domain 4 / RNA polymerase I subunit A N-terminus / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 3 / RNA polymerase, N-terminal / RNA polymerase Rpb2, domain 2 / RNA polymerase, beta subunit, protrusion / RNA polymerase Rpb2, domain 2 / RNA polymerase beta subunit / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / DNA-directed RNA polymerase, insert domain superfamily / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / DNA-directed RNA polymerase, insert domain / RNA polymerases D / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, domain 7 / RNA polymerases beta chain signature. / RNA polymerase Rpb2, domain 6 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain
Similarity search - Domain/homology
DNA-directed RNA polymerase subunit Rpo12 / DNA-directed RNA polymerase subunit Rpo5 / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit / DNA-directed RNA polymerase subunit Rpo1C / DNA-directed RNA polymerase subunit Rpo11 / DNA-directed RNA polymerase, subunit F / DNA-directed RNA polymerase, subunit E / DNA-directed RNA polymerase subunit Rpo10 / DNA-directed RNA polymerase subunit Rpo6 / DNA-directed RNA polymerase subunit Rpo3
Similarity search - Component
Biological speciesThermococcus kodakarensis (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsJun, S.-H. / Hyun, J. / Jeong, H. / Cha, J.S. / Kim, H. / Bartlett, M.S. / Cho, H.-S. / Murakami, K.S.
Funding support Korea, Republic Of, United States, 4items
OrganizationGrant numberCountry
Ministry of Education (Korea)2015R1D1A1A01059097 Korea, Republic Of
Ministry of Science, ICT and Future PlanningNRF-2017M3A9F6029755 Korea, Republic Of
National Institutes of Health/National Institute of General Medical SciencesR35 GM131860 United States
National Institutes of Health/National Institute of Environmental Health SciencesR15 GM083306 United States
CitationJournal: Nat Commun / Year: 2020
Title: Direct binding of TFEα opens DNA binding cleft of RNA polymerase.
Authors: Sung-Hoon Jun / Jaekyung Hyun / Jeong Seok Cha / Hoyoung Kim / Michael S Bartlett / Hyun-Soo Cho / Katsuhiko S Murakami /
Abstract: Opening of the DNA binding cleft of cellular RNA polymerase (RNAP) is necessary for transcription initiation but the underlying molecular mechanism is not known. Here, we report on the cryo-electron ...Opening of the DNA binding cleft of cellular RNA polymerase (RNAP) is necessary for transcription initiation but the underlying molecular mechanism is not known. Here, we report on the cryo-electron microscopy structures of the RNAP, RNAP-TFEα binary, and RNAP-TFEα-promoter DNA ternary complexes from archaea, Thermococcus kodakarensis (Tko). The structures reveal that TFEα bridges the RNAP clamp and stalk domains to open the DNA binding cleft. Positioning of promoter DNA into the cleft closes it while maintaining the TFEα interactions with the RNAP mobile modules. The structures and photo-crosslinking results also suggest that the conserved aromatic residue in the extended winged-helix domain of TFEα interacts with promoter DNA to stabilize the transcription bubble. This study provides a structural basis for the functions of TFEα and elucidates the mechanism by which the DNA binding cleft is opened during transcription initiation in the stalk-containing RNAPs, including archaeal and eukaryotic RNAPs.
History
DepositionJul 6, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 1, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 13, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _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

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Structure visualization

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Assembly

Deposited unit
A: DNA-directed RNA polymerase subunit
B: DNA-directed RNA polymerase subunit beta
C: DNA-directed RNA polymerase subunit A''
D: DNA-directed RNA polymerase subunit D
E: DNA-directed RNA polymerase, subunit E
F: DNA-directed RNA polymerase, subunit F
H: DNA-directed RNA polymerase subunit H
K: DNA-directed RNA polymerase subunit K
L: DNA-directed RNA polymerase subunit L
N: DNA-directed RNA polymerase subunit N
P: DNA-directed RNA polymerase subunit P
hetero molecules


Theoretical massNumber of molelcules
Total (without water)380,40617
Polymers380,05511
Non-polymers3516
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area58600 Å2
ΔGint-393 kcal/mol
Surface area147260 Å2

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Components

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Protein , 1 types, 1 molecules A

#1: Protein DNA-directed RNA polymerase subunit / Polymerase


Mass: 103038.633 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: RpoA'
Source: (natural) Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1) (archaea)
Strain: ATCC BAA-918 / JCM 12380 / KOD1 / References: UniProt: Q5JE33, DNA-directed RNA polymerase

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DNA-directed RNA polymerase subunit ... , 8 types, 8 molecules BCDHKLNP

#2: Protein DNA-directed RNA polymerase subunit beta / Polymerase


Mass: 127468.039 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: RpoB
Source: (natural) Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1) (archaea)
Strain: ATCC BAA-918 / JCM 12380 / KOD1 / References: UniProt: Q5JE32, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit A'' / Polymerase


Mass: 43727.410 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: RpoA''
Source: (natural) Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1) (archaea)
Strain: ATCC BAA-918 / JCM 12380 / KOD1 / References: UniProt: Q5JE34, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit D / Polymerase


Mass: 29429.645 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: RpoD
Source: (natural) Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1) (archaea)
Strain: ATCC BAA-918 / JCM 12380 / KOD1 / References: UniProt: Q5JJF4, DNA-directed RNA polymerase
#7: Protein DNA-directed RNA polymerase subunit H / Polymerase


Mass: 9522.031 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: RpoH
Source: (natural) Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1) (archaea)
Strain: ATCC BAA-918 / JCM 12380 / KOD1 / References: UniProt: Q5JE31, DNA-directed RNA polymerase
#8: Protein DNA-directed RNA polymerase subunit K / Polymerase


Mass: 6286.519 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: RpoK
Source: (natural) Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1) (archaea)
Strain: ATCC BAA-918 / JCM 12380 / KOD1 / References: UniProt: Q5JJD0, DNA-directed RNA polymerase
#9: Protein DNA-directed RNA polymerase subunit L / Polymerase


Mass: 11013.504 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: RpoL
Source: (natural) Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1) (archaea)
Strain: ATCC BAA-918 / JCM 12380 / KOD1 / References: UniProt: Q5JE88, DNA-directed RNA polymerase
#10: Protein DNA-directed RNA polymerase subunit N / Polymerase


Mass: 7601.975 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: RpoN
Source: (natural) Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1) (archaea)
Strain: ATCC BAA-918 / JCM 12380 / KOD1 / References: UniProt: Q5JJC9, DNA-directed RNA polymerase
#11: Protein/peptide DNA-directed RNA polymerase subunit P / Polymerase


Mass: 5553.708 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: RpoP
Source: (natural) Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1) (archaea)
Strain: ATCC BAA-918 / JCM 12380 / KOD1 / References: UniProt: Q5JDM8, DNA-directed RNA polymerase

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DNA-directed RNA polymerase, subunit ... , 2 types, 2 molecules EF

#5: Protein DNA-directed RNA polymerase, subunit E / Polymerase


Mass: 21893.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: RpoE
Source: (gene. exp.) Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1) (archaea)
Strain: ATCC BAA-918 / JCM 12380 / KOD1 / Gene: TK1699 / Production host: Escherichia coli (E. coli) / References: UniProt: Q5JIY4
#6: Protein DNA-directed RNA polymerase, subunit F / Polymerase


Mass: 14519.659 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: RpoF
Source: (gene. exp.) Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1) (archaea)
Strain: ATCC BAA-918 / JCM 12380 / KOD1 / Gene: TK0901 / Production host: Escherichia coli (E. coli) / References: UniProt: Q5JI52

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Non-polymers , 2 types, 6 molecules

#12: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#13: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 5 / 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: RNA polymerase / Type: COMPLEX / Entity ID: #1-#11 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.38 MDa
Source (natural)Organism: Thermococcus kodakarensis KOD1 (archaea)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria) / Strain: BL21
Buffer solutionpH: 8
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 278 K

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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: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 35 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k)

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Processing

EM software
IDNameVersionCategory
12RELION2.1classification
13RELION2.13D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 139242 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT

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