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- PDB-8yf5: Cryo EM structure of Komagataella phaffii Rat1-Rai1-Rtt103 complex -

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Basic information

Entry
Database: PDB / ID: 8yf5
TitleCryo EM structure of Komagataella phaffii Rat1-Rai1-Rtt103 complex
Components
  • 5'-3' exoribonuclease
  • Decapping nuclease
  • Exonuclease Rat1p and Rai1p interacting protein
KeywordsTRANSCRIPTION / transcription termination / RNA polymerase II
Function / homology
Function and homology information


mRNA 5'-diphosphatase activity / NAD-cap decapping / 5'-3' RNA exonuclease activity / nuclease activity / mRNA 3'-end processing / exonuclease activity / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / nuclear-transcribed mRNA catabolic process / RNA polymerase II C-terminal domain binding / DNA-templated transcription termination ...mRNA 5'-diphosphatase activity / NAD-cap decapping / 5'-3' RNA exonuclease activity / nuclease activity / mRNA 3'-end processing / exonuclease activity / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / nuclear-transcribed mRNA catabolic process / RNA polymerase II C-terminal domain binding / DNA-templated transcription termination / mRNA processing / rRNA processing / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / nucleic acid binding / nucleotide binding / RNA binding / nucleus / metal ion binding / 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 / Exonuclease Rat1p and Rai1p interacting protein / 5'-3' exoribonuclease
Similarity search - Component
Biological speciesKomagataella phaffii (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.78 Å
AuthorsYanagisawa, T. / Murayama, Y. / Ehara, H. / Sekine, S.I.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP20H05690 Japan
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis of eukaryotic transcription termination by the Rat1 exonuclease complex.
Authors: Tatsuo Yanagisawa / Yuko Murayama / Haruhiko Ehara / Mie Goto / Mari Aoki / Shun-Ichi Sekine /
Abstract: The 5´-3´ exoribonuclease Rat1/Xrn2 is responsible for the termination of eukaryotic mRNA transcription by RNAPII. Rat1 forms a complex with its partner proteins, Rai1 and Rtt103, and acts as a ...The 5´-3´ exoribonuclease Rat1/Xrn2 is responsible for the termination of eukaryotic mRNA transcription by RNAPII. Rat1 forms a complex with its partner proteins, Rai1 and Rtt103, and acts as a "torpedo" to bind transcribing RNAPII and dissociate DNA/RNA from it. Here we report the cryo-electron microscopy structures of the Rat1-Rai1-Rtt103 complex and three Rat1-Rai1-associated RNAPII complexes (type-1, type-1b, and type-2) from the yeast, Komagataella phaffii. The Rat1-Rai1-Rtt103 structure revealed that Rat1 and Rai1 form a heterotetramer with a single Rtt103 bound between two Rai1 molecules. In the type-1 complex, Rat1-Rai1 forms a heterodimer and binds to the RNA exit site of RNAPII to extract RNA into the Rat1 exonuclease active site. This interaction changes the RNA path in favor of termination (the "pre-termination" state). The type-1b and type-2 complexes have no bound DNA/RNA, likely representing the "post-termination" states. These structures illustrate the termination mechanism of eukaryotic mRNA transcription.
History
DepositionFeb 24, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 25, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: 5'-3' exoribonuclease
B: Decapping nuclease
C: 5'-3' exoribonuclease
D: Decapping nuclease
E: Exonuclease Rat1p and Rai1p interacting protein


Theoretical massNumber of molelcules
Total (without water)362,0945
Polymers362,0945
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein 5'-3' exoribonuclease


Mass: 115314.461 Da / Num. of mol.: 2 / Mutation: D233A, D235A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Komagataella phaffii (fungus) / Gene: RAT1, PP7435_Chr3-0338 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): Rosetta2
References: UniProt: F2QV79, Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters
#2: Protein Decapping nuclease


Mass: 44595.996 Da / Num. of mol.: 2 / Mutation: E213A, D215A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Komagataella phaffii (fungus) / Gene: RAI1, PP7435_Chr1-1057 / Production host: Escherichia coli KRX (bacteria)
References: UniProt: F2QLF5, Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides
#3: Protein Exonuclease Rat1p and Rai1p interacting protein


Mass: 42272.930 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Komagataella phaffii (fungus) / Gene: RTT103, PP7435_Chr1-0928 / Production host: Escherichia coli KRX (bacteria) / References: UniProt: F2QNA8

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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: Komagataella phaffii Rat1-Rai1-Rtt103 complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Komagataella phaffii (fungus)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulphonic acidHEPES1
2100 mMSodium chlorideNaCl1
310 mMpotassium chlorideKCl1
41 mMmagnesium chlorideMgCl21
51 mMTris(2-carboxyethyl)phosphineTCEP1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 75 % / Chamber temperature: 283 K / Details: EMGP2

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1600 nm
Image recordingElectron dose: 61.9 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 3.78 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 77924 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00318933
ELECTRON MICROSCOPYf_angle_d0.52125599
ELECTRON MICROSCOPYf_dihedral_angle_d3.7992479
ELECTRON MICROSCOPYf_chiral_restr0.0412725
ELECTRON MICROSCOPYf_plane_restr0.0033289

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