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- PDB-8q6v: Cryo-EM structure of S. cerevisiae Rai1-Rat1 dimer. -

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Basic information

Entry
Database: PDB / ID: 8q6v
TitleCryo-EM structure of S. cerevisiae Rai1-Rat1 dimer.
Components
  • 5'-3' exoribonuclease 2
  • Decapping nuclease RAI1
KeywordsRNA BINDING PROTEIN / Rai1 nuclease / Rtt103 binding / structured / RNA binding
Function / homology
Function and homology information


RNA polymerase II termination complex / sno(s)RNA processing / positive regulation of termination of RNA polymerase II transcription / RNA NAD+-cap (NAD+-forming) hydrolase activity / termination of RNA polymerase II transcription, poly(A)-coupled / Las1 complex / termination of RNA polymerase II transcription, exosome-dependent / Butyrate Response Factor 1 (BRF1) binds and destabilizes mRNA / Tristetraprolin (TTP, ZFP36) binds and destabilizes mRNA / phosphodiesterase decapping endonuclease activity ...RNA polymerase II termination complex / sno(s)RNA processing / positive regulation of termination of RNA polymerase II transcription / RNA NAD+-cap (NAD+-forming) hydrolase activity / termination of RNA polymerase II transcription, poly(A)-coupled / Las1 complex / termination of RNA polymerase II transcription, exosome-dependent / Butyrate Response Factor 1 (BRF1) binds and destabilizes mRNA / Tristetraprolin (TTP, ZFP36) binds and destabilizes mRNA / phosphodiesterase decapping endonuclease activity / deadenylation-independent decapping of nuclear-transcribed mRNA / mRNA 5'-diphosphatase activity / nuclear polyadenylation-dependent rRNA catabolic process / NAD-cap decapping / 5'-3' RNA exonuclease activity / nuclear mRNA surveillance / maturation of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / negative regulation of transcription elongation by RNA polymerase II / nuclear-transcribed mRNA catabolic process / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / enzyme regulator activity / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / mRNA splicing, via spliceosome / mRNA processing / rRNA processing / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / rRNA binding / nucleotide binding / mRNA binding / mitochondrion / RNA binding / metal ion binding / nucleus / cytosol
Similarity search - Function
5'-3' exoribonuclease type 2 / Xrn1, N-terminal / 5'-3' exoribonuclease / Xrn1, helical domain / XRN 5'-3' exonuclease N-terminus / Xrn1 helical domain / RAI1-like / RAI1-like family / RAI1 like PD-(D/E)XK nuclease
Similarity search - Domain/homology
Decapping nuclease RAI1 / 5'-3' exoribonuclease 2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.23 Å
AuthorsNoskova, N. / Dikunova, A. / Stefl, R.
Funding support Czech Republic, 1items
OrganizationGrant numberCountry
Czech Science Foundation Czech Republic
CitationJournal: Structure / Year: 2025
Title: Assembly of the Xrn2/Rat1-Rai1-Rtt103 termination complexes in mesophilic and thermophilic organisms.
Authors: Alzbeta Dikunova / Nikola Noskova / Jan H Overbeck / Martin Polak / David Stelzig / David Zapletal / Karel Kubicek / Jiri Novacek / Remco Sprangers / Richard Stefl /
Abstract: The 5'-3' exoribonuclease Xrn2, known as Rat1 in yeasts, terminates mRNA transcription by RNA polymerase II (RNAPII). In the torpedo model of termination, the activity of Xrn2/Rat1 is enhanced by ...The 5'-3' exoribonuclease Xrn2, known as Rat1 in yeasts, terminates mRNA transcription by RNA polymerase II (RNAPII). In the torpedo model of termination, the activity of Xrn2/Rat1 is enhanced by Rai1, which is recruited to the termination site by Rtt103, an adaptor protein binding to the RNAPII C-terminal domain (CTD). The overall architecture of the Xrn2/Rat1-Rai1-Rtt103 complex remains unknown. We combined structural biology methods to characterize the torpedo complex from Saccharomyces cerevisiae and Chaetomium thermophilum. Comparison of the structures from these organisms revealed a conserved protein core fold of the subunits, but significant variability in their interaction interfaces. We found that in the mesophile, Rtt103 utilizes an unstructured region to augment a Rai1 β-sheet, while in the thermophile Rtt103 binds to a C-terminal helix of Rai1 via its CTD-interacting domain with an α-helical fold. These different torpedo complex assemblies reflect adaptations to the environment and impact complex recruitment to RNAPII.
History
DepositionAug 15, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 11, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.pdbx_database_id_DOI / _citation_author.name / _em_admin.last_update
Revision 1.2Dec 25, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update
Revision 1.3Feb 19, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Decapping nuclease RAI1
B: 5'-3' exoribonuclease 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)160,6813
Polymers160,6562
Non-polymers241
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Decapping nuclease RAI1 / ScRai1 / NAD-capped RNA hydrolase RAI1 / DeNADding enzyme RAI1 / RAT1-interacting protein


Mass: 44571.445 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RAI1, YGL246C, NRE387 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21-CodonPlus(DE3)-RIPL
References: UniProt: P53063, Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides
#2: Protein 5'-3' exoribonuclease 2


Mass: 116084.930 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RAT1 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21-CodonPlus(DE3)-RIPL / References: UniProt: Q02792
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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: Cryo-EM structure of the S. cerevisiae Rat1-Rai1 complex
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMammonium sulfate(NH4)2SO41
225 mMtris(hydroxymethyl)-aminomethanTris1
32 mMmagnesium sulfateMgSO41
40.1 %glycerolGlycerol1
SpecimenConc.: 1.076 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / 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 FIELD / Nominal defocus max: 3500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
4cryoSPARC4.1.1CTF correction
7Coot0.9.8.7model fitting
8ISOLDEmodel fitting
10Coot0.9.8.7model refinement
11ISOLDEmodel refinement
12cryoSPARC4.1.1initial Euler assignment
13cryoSPARC4.1.1final Euler assignment
14cryoSPARC4.1.1classification
15cryoSPARC4.1.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4408475
3D reconstructionResolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 229534 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: Initial model was obtained with ModelAngelo and then Coot and ISOLDE were used for flexible fitting.
Atomic model building
ID 3D fitting-IDChain-IDDetailsSource nameType
11AModelAngeloOtherin silico model
21BModelAngeloOtherin silico model

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