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- PDB-7ld5: polynucleotide phosphorylase -

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Basic information

Entry
Database: PDB / ID: 7ld5
Titlepolynucleotide phosphorylase
Components
  • Polyribonucleotide nucleotidyltransferase
  • poly-A RNA fragment
KeywordsTRANSFERASE/RNA / phosphorylase polymerase RNA decay / RNA BINDING PROTEIN / TRANSFERASE-RNA complex
Function / homology
Function and homology information


polyribonucleotide nucleotidyltransferase / polyribonucleotide nucleotidyltransferase activity / mRNA catabolic process / RNA processing / 3'-5'-RNA exonuclease activity / magnesium ion binding / RNA binding / cytosol
Similarity search - Function
Guanosine pentaphosphate synthetase I/polyribonucleotide nucleotidyltransferase / Polyribonucleotide nucleotidyltransferase / Polyribonucleotide nucleotidyltransferase, RNA-binding domain / Polyribonucleotide nucleotidyltransferase, RNA-binding domain superfamily / Polyribonucleotide nucleotidyltransferase, RNA binding domain / Exoribonuclease, phosphorolytic domain 1 / PNPase/RNase PH domain superfamily / Exoribonuclease, PH domain 2 superfamily / 3' exoribonuclease family, domain 1 / KH domain ...Guanosine pentaphosphate synthetase I/polyribonucleotide nucleotidyltransferase / Polyribonucleotide nucleotidyltransferase / Polyribonucleotide nucleotidyltransferase, RNA-binding domain / Polyribonucleotide nucleotidyltransferase, RNA-binding domain superfamily / Polyribonucleotide nucleotidyltransferase, RNA binding domain / Exoribonuclease, phosphorolytic domain 1 / PNPase/RNase PH domain superfamily / Exoribonuclease, PH domain 2 superfamily / 3' exoribonuclease family, domain 1 / KH domain / K Homology domain, type 1 / Type-1 KH domain profile. / K Homology domain, type 1 superfamily / S1 domain profile. / Ribosomal protein S1-like RNA-binding domain / S1 RNA binding domain / S1 domain / K Homology domain / K homology RNA-binding domain / Ribosomal protein S5 domain 2-type fold / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
RNA / Polyribonucleotide nucleotidyltransferase
Similarity search - Component
Biological speciesMycolicibacterium smegmatis (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.07 Å
AuthorsGoldgur, Y. / Shuman, S. / De La Cruz, M.J. / Ghosh, S. / Unciuleac, M.-C.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35-GM126945 United States
CitationJournal: RNA / Year: 2021
Title: Structure and mechanism of polynucleotide phosphorylase.
Authors: Mihaela-Carmen Unciuleac / Shreya Ghosh / M Jason de la Cruz / Yehuda Goldgur / Stewart Shuman
Abstract: Polynucleotide phosphorylase (PNPase) catalyzes stepwise phosphorolysis of the 3'-terminal phosphodiesters of RNA chains to yield nucleoside diphosphate products. In the reverse reaction PNPase acts ...Polynucleotide phosphorylase (PNPase) catalyzes stepwise phosphorolysis of the 3'-terminal phosphodiesters of RNA chains to yield nucleoside diphosphate products. In the reverse reaction PNPase acts as a polymerase, using NDPs as substrates to add NMPs to the 3'-OH terminus of RNA chains while expelling inorganic phosphate. The apparent essentiality of PNPase for growth of militates for mycobacterial PNPase as a potential drug target. A cryo-EM structure of PNPase (MsmPNPase) reveals a characteristic ring-shaped homotrimer in which each protomer consists of two RNase PH-like domains and an intervening α-helical module on the inferior surface of the ring. The C-terminal KH and S1 domains, which impart RNA specificity to MsmPNPase, are on the opposite face of the core ring and are conformationally mobile. Single particle reconstructions of MsmPNPase in the act of poly(A) synthesis highlight a 3'-terminal (rA)4 oligonucleotide and two magnesium ions in the active site and an adenine nucleobase in the central tunnel. We identify amino acids that engage the 3' segment of the RNA chain (Phe68, Arg105, Arg112, Arg430, Arg431) and the two metal ions (Asp526, Asp532, Gln546, Asp548) and we infer those that bind inorganic phosphate (Thr470, Ser471, His435, Lys534). Alanine mutagenesis pinpointed RNA and phosphate contacts as essential (Arg105, Arg431, Lys534, Thr470+Ser471), important (Arg112, Arg430), or unimportant (Phe68) for PNPase activity. Severe phosphorolysis and polymerase defects accompanying alanine mutations of the enzymic metal ligands suggest a two-metal mechanism of catalysis by MsmPNPase.
History
DepositionJan 12, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 30, 2021Provider: repository / Type: Initial release
Revision 1.1Nov 30, 2022Group: Database references / Category: citation / database_2
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2May 29, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Assembly

Deposited unit
A: Polyribonucleotide nucleotidyltransferase
B: Polyribonucleotide nucleotidyltransferase
C: Polyribonucleotide nucleotidyltransferase
D: poly-A RNA fragment
E: poly-A RNA fragment
F: poly-A RNA fragment
hetero molecules


Theoretical massNumber of molelcules
Total (without water)259,84312
Polymers259,6976
Non-polymers1466
Water00
1


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

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Components

#1: Protein Polyribonucleotide nucleotidyltransferase / Polynucleotide phosphorylase / PNPase


Mass: 83647.703 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis (bacteria) / Gene: pnp, gpsI, MSMEG_2656, MSMEI_2593 / Production host: Escherichia coli (E. coli)
References: UniProt: A0QVQ5, polyribonucleotide nucleotidyltransferase
#2: RNA chain poly-A RNA fragment


Mass: 2917.895 Da / Num. of mol.: 3 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN
Sequence detailsAuthors state that the exact length of RNA fragment is unknown.

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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: Polynucleotide phosphorylase complex in poly(A) synthesis
Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Mycolicibacterium smegmatis (bacteria)1772
21synthetic construct (others)32630
Source (recombinant)Organism: Escherichia coli (E. coli)
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

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 FIELD
Image recordingElectron dose: 53 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameVersionCategory
4CTFFIND4.1CTF correction
7PHENIX1.17.1-3660model fitting
9cryoSPARC2.16.1initial Euler assignment
10cryoSPARC2.16.1final Euler assignment
13PHENIX1.17.1-3660model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.07 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 890249 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00613788
ELECTRON MICROSCOPYf_angle_d0.63818774
ELECTRON MICROSCOPYf_dihedral_angle_d5.0962046
ELECTRON MICROSCOPYf_chiral_restr0.0482184
ELECTRON MICROSCOPYf_plane_restr0.0042439

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