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- PDB-9dso: CRYO-EM STRUCTURE OF SACCHAROMYCES CEREVISIAE RAT1-RAI1-RTT103 COMPLEX -

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Basic information

Entry
Database: PDB / ID: 9dso
TitleCRYO-EM STRUCTURE OF SACCHAROMYCES CEREVISIAE RAT1-RAI1-RTT103 COMPLEX
Components
  • 5'-3' exoribonuclease 2
  • Decapping nuclease RAI1
  • Regulator of Ty1 transposition protein 103
KeywordsTRANSCRIPTION / Nuclease / Complex / Transcription termination
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 / RNA polymerase II C-terminal domain phosphoserine binding / nuclear polyadenylation-dependent rRNA catabolic process / NAD-cap decapping / 5'-3' RNA exonuclease activity / nuclear mRNA surveillance / transposable element silencing / mRNA 3'-end processing / maturation of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / RNA polymerase II transcribes snRNA genes / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / RNA polymerase II complex binding / 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 / site of double-strand break / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / rRNA binding / nucleotide binding / mRNA binding / chromatin / mitochondrion / DNA binding / 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 ...: / 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 / CID domain / RPR / CID domain / CID domain profile. / ENTH/VHS
Similarity search - Domain/homology
Decapping nuclease RAI1 / 5'-3' exoribonuclease 2 / Regulator of Ty1 transposition protein 103
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.85 Å
AuthorsChu, H.F. / Tong, L.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118093 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24 GM129539 United States
Simons FoundationSF349247 United States
CitationJournal: Nat Commun / Year: 2025
Title: Molecular basis for the interaction between Saccharomyces cerevisiae Rtt103 and the Rat1-Rai1 complex.
Authors: Hsu-Feng Chu / Liang Tong /
Abstract: The Rat1 5'-3' exoribonuclease together with its partner Rai1 have important roles in Saccharomyces cerevisiae RNA polymerase II transcription termination. Rtt103 copurifies with Rat1-Rai1 in S. ...The Rat1 5'-3' exoribonuclease together with its partner Rai1 have important roles in Saccharomyces cerevisiae RNA polymerase II transcription termination. Rtt103 copurifies with Rat1-Rai1 in S. cerevisiae, but its mechanism of interaction with them is not known. We report here the cryo-EM structure of the S. cerevisiae Rat1-Rai1-Rtt103 ternary complex at 2.9 Å resolution. We found that a short segment of Rtt103 is in close contact with Rai1, while the rest of Rtt103, including its RNA polymerase II C-terminal domain interaction domain, shows no interactions with Rai1 or Rat1. This is in contrast to the observations on the Komagataella phaffii Rat1-Rai1-Rtt103 complex, where only the RNA polymerase II C-terminal domain interaction domain of Rtt103 has contacts with Rai1. Our structure reveals that S. cerevisiae Rtt103 Pro261 and Tyr263 have important contacts with Rai1, and we show that the P261G/Y263A mutation of Rtt103 blocks the interaction with Rat1-Rai1. Our structure suggests that, in yeast, this segment of Rtt103, which we have named the Rai1 interaction segment, likely helps the recruitment of Rat1-Rai1 to RNA polymerase II for termination.
History
DepositionSep 28, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 16, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: 5'-3' exoribonuclease 2
B: Decapping nuclease RAI1
C: Regulator of Ty1 transposition protein 103
hetero molecules


Theoretical massNumber of molelcules
Total (without water)207,5254
Polymers207,5013
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 5'-3' exoribonuclease 2 / Ribonucleic acid-trafficking protein 1 / p116


Mass: 114204.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RAT1, HKE1, TAP1, YOR048C / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: Q02792, Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters
#2: 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: Trichoplusia ni (cabbage looper)
References: UniProt: P53063, Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides
#3: Protein Regulator of Ty1 transposition protein 103


Mass: 48725.473 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RTT103, YDR289C / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q05543
#4: 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: Rat1-Rai1-Rtt103 complex / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
2200 mMsodium cholorideNaCl1
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 293 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 49.27 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.5particle selection
2Leginonimage acquisition
4cryoSPARC4.5CTF correction
7UCSF ChimeraX1.8model fitting
8Coot0.9.8model fitting
10PHENIX1.20.1model refinement
11cryoSPARC4.5initial Euler assignment
12cryoSPARC4.5final Euler assignment
13cryoSPARC4.5classification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 614995
3D reconstructionResolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 552608 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0049197
ELECTRON MICROSCOPYf_angle_d0.51712461
ELECTRON MICROSCOPYf_dihedral_angle_d4.141211
ELECTRON MICROSCOPYf_chiral_restr0.0431345
ELECTRON MICROSCOPYf_plane_restr0.0041617

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