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- PDB-8a8f: Crystal structure of Glc7 phosphatase in complex with the regulat... -

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Basic information

Entry
Database: PDB / ID: 8a8f
TitleCrystal structure of Glc7 phosphatase in complex with the regulatory region of Ref2
Components
  • RNA end formation protein 2
  • Serine/threonine-protein phosphatase PP1-2
KeywordsGENE REGULATION / Ser/Thr phosphatase / regulatory subunit / RNA / transcription termination
Function / homology
Function and homology information


regulation of cell budding / regulation of protein localization to cell division site involved in cytokinesis / cellular bud neck septin collar / mating projection base / positive regulation of mitotic actomyosin contractile ring assembly / regulation of cellular response to glucose starvation / Resolution of Sister Chromatid Cohesion / positive regulation of clathrin-dependent endocytosis / termination of RNA polymerase II transcription, poly(A)-coupled / sno(s)RNA 3'-end processing ...regulation of cell budding / regulation of protein localization to cell division site involved in cytokinesis / cellular bud neck septin collar / mating projection base / positive regulation of mitotic actomyosin contractile ring assembly / regulation of cellular response to glucose starvation / Resolution of Sister Chromatid Cohesion / positive regulation of clathrin-dependent endocytosis / termination of RNA polymerase II transcription, poly(A)-coupled / sno(s)RNA 3'-end processing / regulation of glycogen biosynthetic process / chitin biosynthetic process / ascospore formation / cellular bud site selection / regulation of glycogen metabolic process / positive regulation of exit from mitosis / protein phosphatase type 1 complex / incipient cellular bud site / intracellular monoatomic ion homeostasis / mRNA cleavage and polyadenylation specificity factor complex / negative regulation of actin filament polymerization / cellular bud neck / DNA replication checkpoint signaling / protein localization to kinetochore / spindle pole body / mRNA 3'-end processing / protein serine/threonine phosphatase activity / glycogen metabolic process / histone H2AXS140 phosphatase activity / RNA polymerase II CTD heptapeptide repeat Y1 phosphatase activity / RNA polymerase II CTD heptapeptide repeat T4 phosphatase activity / RNA polymerase II CTD heptapeptide repeat S2 phosphatase activity / RNA polymerase II CTD heptapeptide repeat S5 phosphatase activity / RNA polymerase II CTD heptapeptide repeat S7 phosphatase activity / MAP kinase serine/threonine phosphatase activity / calmodulin-dependent protein phosphatase activity / myosin phosphatase activity / protein-serine/threonine phosphatase / cell division site / mitotic spindle assembly checkpoint signaling / replication fork processing / chromosome organization / response to unfolded protein / telomere maintenance / regulation of mitotic cell cycle / DNA damage checkpoint signaling / meiotic cell cycle / chromosome segregation / kinetochore / mRNA processing / regulation of cell shape / response to heat / regulation of cell cycle / chromatin binding / nucleolus / positive regulation of transcription by RNA polymerase II / RNA binding / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Serine-threonine protein phosphatase, N-terminal / : / Serine-threonine protein phosphatase N-terminal domain / Serine/threonine specific protein phosphatases signature. / Protein phosphatase 2A homologues, catalytic domain. / Serine/threonine-specific protein phosphatase/bis(5-nucleosyl)-tetraphosphatase / Calcineurin-like phosphoesterase domain, ApaH type / Calcineurin-like phosphoesterase / Metallo-dependent phosphatase-like
Similarity search - Domain/homology
: / PHOSPHATE ION / Serine/threonine-protein phosphatase PP1-2 / RNA end formation protein 2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.85 Å
AuthorsCarminati, M. / Manav, C.M. / Bellini, D. / Passmore, L.A.
Funding supportEuropean Union, 2items
OrganizationGrant numberCountry
European Union (EU)725685European Union
European Molecular Biology Organization (EMBO)556-2018European Union
CitationJournal: Mol Cell / Year: 2023
Title: A direct interaction between CPF and RNA Pol II links RNA 3' end processing to transcription.
Authors: Manuel Carminati / Juan B Rodríguez-Molina / M Cemre Manav / Dom Bellini / Lori A Passmore /
Abstract: Transcription termination by RNA polymerase II (RNA Pol II) is linked to RNA 3' end processing by the cleavage and polyadenylation factor (CPF or CPSF). CPF contains endonuclease, poly(A) polymerase, ...Transcription termination by RNA polymerase II (RNA Pol II) is linked to RNA 3' end processing by the cleavage and polyadenylation factor (CPF or CPSF). CPF contains endonuclease, poly(A) polymerase, and protein phosphatase activities, which cleave and polyadenylate pre-mRNAs and dephosphorylate RNA Pol II to control transcription. Exactly how the RNA 3' end processing machinery is coupled to transcription remains unclear. Here, we combine in vitro reconstitution, structural studies, and genome-wide analyses to show that yeast CPF physically and functionally interacts with RNA Pol II. Surprisingly, CPF-mediated dephosphorylation promotes the formation of an RNA Pol II stalk-to-stalk homodimer in vitro. This dimer is compatible with transcription but not with the binding of transcription elongation factors. Disruption of the dimerization interface in cells causes transcription defects, including altered RNA Pol II abundance on protein-coding genes, tRNA genes, and intergenic regions. We hypothesize that RNA Pol II dimerization may provide a mechanistic basis for the allosteric model of transcription termination.
History
DepositionJun 22, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 29, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 12, 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Serine/threonine-protein phosphatase PP1-2
B: RNA end formation protein 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)44,7425
Polymers44,5372
Non-polymers2053
Water2,180121
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)90.230, 90.230, 101.140
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number152
Space group name H-MP3121

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Components

#1: Protein Serine/threonine-protein phosphatase PP1-2


Mass: 36036.344 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The Glc7 C-terminus is covalently linked to chain B (Ref2 348-405)
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Strain: ATCC 204508 / S288c / Gene: GLC7, CID1, DIS2, YER133W / Plasmid: pET24a
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P32598, protein-serine/threonine phosphatase
#2: Protein RNA end formation protein 2


Mass: 8500.543 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: To trap the Glc7 substrate in the active site, two RNA Pol II CTD repeats were fused C-terminally of Ref2 348-405. The substrate was not visible in the crystal structure.
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Strain: ATCC 204508 / S288c / Gene: REF2, YDR195W, YD9346.06
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P42073
#3: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn
#4: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 121 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.67 Å3/Da / Density % sol: 53.91 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8
Details: 40 % (v/v) 1,4-Butanediol and 0.1 M Tris-HCl pH 8.5
PH range: 8.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04 / Wavelength: 0.9795 Å
DetectorType: DECTRIS EIGER2 XE 16M / Detector: PIXEL / Date: Dec 16, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 1.85→45.12 Å / Num. obs: 41059 / % possible obs: 99.96 % / Redundancy: 10 % / CC1/2: 1 / Rmerge(I) obs: 0.04671 / Net I/σ(I): 20.46
Reflection shellResolution: 1.85→1.917 Å / Redundancy: 10 % / Rmerge(I) obs: 1.224 / Mean I/σ(I) obs: 1.84 / Num. unique obs: 4028 / CC1/2: 0.9 / % possible all: 98.82

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 7QWJ

7qwj


Resolution: 1.85→45.12 Å / SU ML: 0.24 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 28.08 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2205 2090 5.1 %
Rwork0.192 38883 -
obs0.1935 40973 99.69 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 122.67 Å2 / Biso mean: 54.6302 Å2 / Biso min: 33.34 Å2
Refinement stepCycle: final / Resolution: 1.85→45.12 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2851 0 7 121 2979
Biso mean--37.78 54.99 -
Num. residues----354
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 15

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.85-1.890.42241410.31432533267498
1.89-1.940.35721320.28652542267499
1.94-1.990.33031330.265225702703100
1.99-2.050.27961410.246325692710100
2.05-2.120.29521150.23625772692100
2.12-2.190.30851210.230825942715100
2.19-2.280.26961690.21325052674100
2.28-2.390.25371440.211525842728100
2.39-2.510.26371400.211225892729100
2.51-2.670.26581160.216725982714100
2.67-2.870.23491350.22626062741100
2.88-3.160.23341140.212326402754100
3.16-3.620.2581660.20125872753100
3.62-4.560.1841620.168626382800100
4.56-45.120.17451610.155427512912100

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