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- PDB-8yfr: Cryo EM structure of Komagataella phaffii Rat1-Rai1 complex bound... -

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Basic information

Entry
Database: PDB / ID: 8yfr
TitleCryo EM structure of Komagataella phaffii Rat1-Rai1 complex bound within the RNAPII cleft
Components
  • (DNA-directed RNA polymerase ...) x 3
  • (DNA-directed RNA polymerases I, II, and III subunit ...) x 2
  • (RNA polymerase II ...) x 4
  • (RNA polymerase subunit ABC10- ...) x 2
  • 5'-3' exoribonuclease
  • Decapping nuclease
  • RNA polymerase subunit ABC23, common to RNA polymerases I, II, and III
KeywordsTRANSCRIPTION / transcription termination / RNA polymerase II
Function / homology
Function and homology information


mRNA 5'-diphosphatase activity / regulation of septum digestion after cytokinesis / NAD-cap decapping / siRNA-mediated pericentric heterochromatin formation / 5'-3' RNA exonuclease activity / nuclease activity / RPB4-RPB7 complex / nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / termination of RNA polymerase II transcription / positive regulation of nuclear-transcribed mRNA poly(A) tail shortening ...mRNA 5'-diphosphatase activity / regulation of septum digestion after cytokinesis / NAD-cap decapping / siRNA-mediated pericentric heterochromatin formation / 5'-3' RNA exonuclease activity / nuclease activity / RPB4-RPB7 complex / nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / termination of RNA polymerase II transcription / positive regulation of nuclear-transcribed mRNA poly(A) tail shortening / termination of RNA polymerase III transcription / termination of RNA polymerase I transcription / transcription initiation at RNA polymerase III promoter / transcription initiation at RNA polymerase I promoter / maintenance of transcriptional fidelity during transcription elongation by RNA polymerase II / nuclear-transcribed mRNA catabolic process / positive regulation of translational initiation / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / RNA polymerase I complex / tRNA transcription by RNA polymerase III / RNA polymerase III complex / transcription elongation by RNA polymerase I / pericentric heterochromatin / transcription-coupled nucleotide-excision repair / RNA polymerase II, core complex / : / translation initiation factor binding / DNA-directed RNA polymerase activity / transcription initiation at RNA polymerase II promoter / transcription elongation by RNA polymerase II / P-body / DNA-templated transcription termination / ribonucleoside binding / : / : / : / : / : / : / DNA-directed RNA polymerase / mRNA processing / rRNA processing / single-stranded DNA binding / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / nucleic acid binding / single-stranded RNA binding / protein dimerization activity / RNA-directed RNA polymerase activity / nucleotide binding / nucleolus / DNA binding / RNA binding / zinc ion 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 / DNA-directed RNA polymerase II subunit Rpb4-like ...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 / DNA-directed RNA polymerase II subunit Rpb4-like / RNA polymerase Rpb1 C-terminal repeat / RNA polymerase II, heptapeptide repeat, eukaryotic / Eukaryotic RNA polymerase II heptapeptide repeat. / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb4/RPC9, core / DNA-directed RNA-polymerase II subunit / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 7 superfamily / RNA polymerase Rpb1, domain 7 / Pol II subunit B9, C-terminal zinc ribbon / RNA polymerase RBP11 / Rpb4/RPC9 superfamily / S1 domain profile. / RNA polymerase subunit Rpb4/RPC9 / RNA polymerase Rpb4 / HRDC-like superfamily / RNA polymerase Rpb7-like , N-terminal / RNA polymerase Rpb7-like, N-terminal domain superfamily / RNA polymerase subunit Rpb7-like / SHS2 domain found in N terminus of Rpb7p/Rpc25p/MJ0397 / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / DNA-directed RNA polymerase, M/15kDa subunit / RNA polymerases M/15 Kd subunit / RNA polymerase subunit 9 / DNA-directed RNA polymerase M, 15kDa subunit, conserved site / RNA polymerases M / 15 Kd subunits signature. / DNA-directed RNA polymerase subunit/transcription factor S / : / RNA polymerase, Rpb8 / DNA-directed RNA polymerases I, II, and III subunit RPABC4 / RNA polymerase Rpb8 / RNA polymerase subunit 8 / RNA polymerase, Rpb5, N-terminal / RNA polymerase Rpb5, N-terminal domain superfamily / RNA polymerase Rpb5, N-terminal domain / DNA-directed RNA polymerase, subunit RPB6 / DNA-directed RNA polymerase subunit RPABC5/Rpb10 / RNA polymerases, subunit N, zinc binding site / RNA polymerase subunit RPB10 / RNA polymerases N / 8 kDa subunit / RNA polymerases N / 8 Kd subunits signature. / DNA directed RNA polymerase, 7 kDa subunit / RNA polymerase archaeal subunit P/eukaryotic subunit RPABC4 / RNA polymerase, subunit H/Rpb5, conserved site / RNA polymerases H / 23 Kd subunits signature. / RNA polymerase subunit CX / DNA-directed RNA polymerase, 30-40kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo3/Rpb3/RPAC1 / RNA polymerases D / 30 to 40 Kd subunits signature. / DNA-directed RNA polymerase Rpb11, 13-16kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo11 / RNA polymerases L / 13 to 16 Kd subunits signature. / Zinc finger, TFIIS-type / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / Transcription factor S-II (TFIIS) / Zinc finger TFIIS-type profile. / C2C2 Zinc finger / DNA-directed RNA polymerase, RBP11-like dimerisation domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase, subunit H/Rpb5 C-terminal / DNA-directed RNA polymerase subunit Rpo5/Rpb5 / RPB5-like RNA polymerase subunit superfamily / RNA polymerase Rpb5, C-terminal domain / Archaeal Rpo6/eukaryotic RPB6 RNA polymerase subunit / DNA-directed RNA polymerase, 14-18kDa subunit, conserved site / RNA polymerases K / 14 to 18 Kd subunits signature. / S1 RNA binding domain / S1 domain / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, beta subunit, protrusion
Similarity search - Domain/homology
DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerases I, II, and III subunit RPABC5 / RNA polymerase subunit ABC23, common to RNA polymerases I, II, and III / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / RNA polymerase II subunit B32 / DNA-directed RNA polymerases I, II, and III subunit RPABC1 / RNA polymerase II subunit B12.5 / DNA-directed RNA polymerase subunit / RNA polymerase II third largest subunit B44, part of central core / DNA-directed RNA polymerase subunit ...DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerases I, II, and III subunit RPABC5 / RNA polymerase subunit ABC23, common to RNA polymerases I, II, and III / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / RNA polymerase II subunit B32 / DNA-directed RNA polymerases I, II, and III subunit RPABC1 / RNA polymerase II subunit B12.5 / DNA-directed RNA polymerase subunit / RNA polymerase II third largest subunit B44, part of central core / DNA-directed RNA polymerase subunit / Decapping nuclease / RNA polymerase subunit ABC10-alpha / DNA-directed RNA polymerase subunit / 5'-3' exoribonuclease
Similarity search - Component
Biological speciesKomagataella phaffii (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsMurayama, Y. / Yanagisawa, T. / Ehara, H. / Sekine, S.
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 25, 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: DNA-directed RNA polymerase subunit
B: DNA-directed RNA polymerase subunit beta
C: RNA polymerase II third largest subunit B44, part of central core
D: RNA polymerase II subunit B32
E: DNA-directed RNA polymerases I, II, and III subunit RPABC1
F: RNA polymerase subunit ABC23, common to RNA polymerases I, II, and III
G: RNA polymerase II subunit
H: DNA-directed RNA polymerases I, II, and III subunit RPABC3
I: DNA-directed RNA polymerase subunit
J: RNA polymerase subunit ABC10-beta, common to RNA polymerases I, II, and III
K: RNA polymerase II subunit B12.5
L: RNA polymerase subunit ABC10-alpha
R: 5'-3' exoribonuclease
S: Decapping nuclease
hetero molecules


Theoretical massNumber of molelcules
Total (without water)670,91323
Polymers670,36614
Non-polymers5489
Water00
1


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

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Components

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DNA-directed RNA polymerase ... , 3 types, 3 molecules ABI

#1: Protein DNA-directed RNA polymerase subunit


Mass: 194107.422 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Komagataella phaffii (fungus) / References: UniProt: C4R4Y0, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta


Mass: 139746.094 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Komagataella phaffii (fungus) / References: UniProt: C4QZQ7, DNA-directed RNA polymerase
#9: Protein DNA-directed RNA polymerase subunit


Mass: 13612.320 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Komagataella phaffii (fungus) / References: UniProt: F2QPE6

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RNA polymerase II ... , 4 types, 4 molecules CDGK

#3: Protein RNA polymerase II third largest subunit B44, part of central core


Mass: 34216.293 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Komagataella phaffii (fungus) / References: UniProt: C4R7L2
#4: Protein RNA polymerase II subunit B32


Mass: 20622.980 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Komagataella phaffii (fungus) / References: UniProt: C4R2U9
#7: Protein RNA polymerase II subunit


Mass: 18802.625 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Komagataella phaffii (fungus) / References: UniProt: C4R9A1
#11: Protein RNA polymerase II subunit B12.5


Mass: 13832.896 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Komagataella phaffii (fungus) / References: UniProt: C4R3Z5

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DNA-directed RNA polymerases I, II, and III subunit ... , 2 types, 2 molecules EH

#5: Protein DNA-directed RNA polymerases I, II, and III subunit RPABC1


Mass: 24962.680 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Komagataella phaffii (fungus) / References: UniProt: C4R3P8
#8: Protein DNA-directed RNA polymerases I, II, and III subunit RPABC3


Mass: 16249.220 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Komagataella phaffii (fungus) / References: UniProt: C4R273

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Protein , 3 types, 3 molecules FRS

#6: Protein RNA polymerase subunit ABC23, common to RNA polymerases I, II, and III


Mass: 17803.588 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Komagataella phaffii (fungus) / References: UniProt: C4R1V1
#13: Protein 5'-3' exoribonuclease


Mass: 115397.555 Da / Num. of mol.: 1 / Mutation: E203Q, E205Q, D233N, D235N, D330N
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)
References: UniProt: F2QV79, Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters
#14: Protein Decapping nuclease


Mass: 44595.996 Da / Num. of mol.: 1 / 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 BL21(DE3) (bacteria)
References: UniProt: F2QLF5, Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides

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RNA polymerase subunit ABC10- ... , 2 types, 2 molecules JL

#10: Protein RNA polymerase subunit ABC10-beta, common to RNA polymerases I, II, and III


Mass: 8554.064 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Komagataella phaffii (fungus) / References: UniProt: C4R009
#12: Protein RNA polymerase subunit ABC10-alpha


Mass: 7862.048 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Komagataella phaffii (fungus) / References: UniProt: F2QMI1

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Non-polymers , 2 types, 9 molecules

#15: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Zn
#16: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN

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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 complex bound within the RNAPII cleft
Type: COMPLEX / Entity ID: #1-#14 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Komagataella phaffii (fungus)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: 53.3 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 35181 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00340127
ELECTRON MICROSCOPYf_angle_d0.53354189
ELECTRON MICROSCOPYf_dihedral_angle_d3.9095360
ELECTRON MICROSCOPYf_chiral_restr0.0435980
ELECTRON MICROSCOPYf_plane_restr0.0037014

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