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- PDB-7zgr: Polymerase module of yeast CPF in complex with Mpe1, the yPIM of ... -

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Basic information

Entry
Database: PDB / ID: 7zgr
TitlePolymerase module of yeast CPF in complex with Mpe1, the yPIM of Cft2 and the pre-cleaved CYC1 RNA
Components
  • Cleavage factor two protein 2
  • MPE1 isoform 1
  • Polyadenylation factor subunit 2
  • Protein CFT1
  • mRNA 3'-end-processing protein YTH1
  • pre-cleaved CYC1
KeywordsRNA BINDING PROTEIN / CPF / 3'-end processing
Function / homology
Function and homology information


Processing of Intronless Pre-mRNAs / termination of RNA polymerase II transcription, poly(A)-coupled / intracellular organelle / mRNA cleavage and polyadenylation specificity factor complex / mRNA 3'-end processing / : / termination of RNA polymerase II transcription / mRNA processing / ubiquitin protein ligase activity / nucleic acid binding ...Processing of Intronless Pre-mRNAs / termination of RNA polymerase II transcription, poly(A)-coupled / intracellular organelle / mRNA cleavage and polyadenylation specificity factor complex / mRNA 3'-end processing / : / termination of RNA polymerase II transcription / mRNA processing / ubiquitin protein ligase activity / nucleic acid binding / mitochondrion / RNA binding / zinc ion binding / metal ion binding / nucleus
Similarity search - Function
E3 ubiquitin-protein ligase RBBP6 family / DWNN domain / DWNN domain / DWNN domain profile. / DWNN / Cleavage and polyadenylation specificity factor 2, C-terminal / CPSF2, metallo-hydrolase domain / Cleavage and polyadenylation factor 2 C-terminal / Cleavage and polyadenylation specificity factor subunit 2 / CPSF complex subunit CPSF4-like ...E3 ubiquitin-protein ligase RBBP6 family / DWNN domain / DWNN domain / DWNN domain profile. / DWNN / Cleavage and polyadenylation specificity factor 2, C-terminal / CPSF2, metallo-hydrolase domain / Cleavage and polyadenylation factor 2 C-terminal / Cleavage and polyadenylation specificity factor subunit 2 / CPSF complex subunit CPSF4-like / RNA-binding, Nab2-type zinc finger / Pre-mRNA 3' end processing protein Pfs2-like / Metallo-beta-lactamase superfamily domain / Beta-Casp domain / Beta-Casp domain / Zinc finger C-x8-C-x5-C-x3-H type (and similar) / Zinc finger, CCCH-type superfamily / zinc finger / Zinc finger, CCCH-type / Zinc finger C3H1-type profile. / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / CPSF A subunit region / Metallo-beta-lactamase / Ribonuclease Z/Hydroxyacylglutathione hydrolase-like / zinc finger / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile. / Zinc finger, RING/FYVE/PHD-type / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / MPE1 isoform 1 / mRNA 3'-end-processing protein / Polyadenylation factor subunit 2 / Protein CFT1 / Cleavage factor two protein 2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsRodriguez-Molina, J.B. / Passmore, L.A.
Funding supportEuropean Union, United Kingdom, 2items
OrganizationGrant numberCountry
European Research Council (ERC)725685European Union
Medical Research Council (MRC, United Kingdom)MC_U105192715 United Kingdom
CitationJournal: Mol Cell / Year: 2022
Title: Mpe1 senses the binding of pre-mRNA and controls 3' end processing by CPF.
Authors: Juan B Rodríguez-Molina / Francis J O'Reilly / Holly Fagarasan / Eleanor Sheekey / Sarah Maslen / J Mark Skehel / Juri Rappsilber / Lori A Passmore /
Abstract: Most eukaryotic messenger RNAs (mRNAs) are processed at their 3' end by the cleavage and polyadenylation specificity factor (CPF/CPSF). CPF mediates the endonucleolytic cleavage of the pre-mRNA and ...Most eukaryotic messenger RNAs (mRNAs) are processed at their 3' end by the cleavage and polyadenylation specificity factor (CPF/CPSF). CPF mediates the endonucleolytic cleavage of the pre-mRNA and addition of a polyadenosine (poly(A)) tail, which together define the 3' end of the mature transcript. The activation of CPF is highly regulated to maintain the fidelity of RNA processing. Here, using cryo-EM of yeast CPF, we show that the Mpe1 subunit directly contacts the polyadenylation signal sequence in nascent pre-mRNA. The region of Mpe1 that contacts RNA also promotes the activation of CPF endonuclease activity and controls polyadenylation. The Cft2 subunit of CPF antagonizes the RNA-stabilized configuration of Mpe1. In vivo, the depletion or mutation of Mpe1 leads to widespread defects in transcription termination by RNA polymerase II, resulting in transcription interference on neighboring genes. Together, our data suggest that Mpe1 plays a major role in accurate 3' end processing, activating CPF, and ensuring timely transcription termination.
History
DepositionApr 4, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 25, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 1, 2022Group: 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.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Jul 20, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Protein CFT1
B: mRNA 3'-end-processing protein YTH1
C: Cleavage factor two protein 2
D: Polyadenylation factor subunit 2
E: pre-cleaved CYC1
F: MPE1 isoform 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)375,5488
Polymers375,4176
Non-polymers1312
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 5 types, 5 molecules ABCDF

#1: Protein Protein CFT1 / Cleavage factor two protein 1


Mass: 153577.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Strain: ATCC 204508 / S288c / Gene: CFT1, YHH1, YDR301W / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q06632
#2: Protein mRNA 3'-end-processing protein YTH1


Mass: 24594.498 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: YTH1, GI527_G0006153 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A0A6A5Q2R8
#3: Protein Cleavage factor two protein 2 / 105 kDa protein associated with polyadenylation factor I


Mass: 80993.055 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Strain: ATCC 204508 / S288c / Gene: CFT2, YDH1, YLR115W, L2946 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q12102
#4: Protein Polyadenylation factor subunit 2


Mass: 53211.117 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: PFS2, GI527_G0004795 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A0A6A5Q543
#6: Protein MPE1 isoform 1


Mass: 49711.848 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MPE1, GI527_G0003582 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A0A6A5PV64

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RNA chain / Non-polymers , 2 types, 3 molecules E

#5: RNA chain pre-cleaved CYC1


Mass: 13329.796 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast)
#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / 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: polymerase module-Mpe1-yPIM-RNA / Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8 / Details: 20 mM HEPES pH 8, 50 mM NaCl, 0.5 mM TCEP
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 3100 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 19524
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPUimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 13905256
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 846349 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-ID
17ZGP

7zgp

1
21ZGQ

1zgq

1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00314830
ELECTRON MICROSCOPYf_angle_d0.53720113
ELECTRON MICROSCOPYf_dihedral_angle_d15.5165489
ELECTRON MICROSCOPYf_chiral_restr0.0442273
ELECTRON MICROSCOPYf_plane_restr0.0032561

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