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- PDB-8h5b: The cryo-EM structure of nuclear transport receptor Kap114p compl... -

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Basic information

Entry
Database: PDB / ID: 8h5b
TitleThe cryo-EM structure of nuclear transport receptor Kap114p complex with yeast TATA-box binding protein
Components
  • Importin subunit beta-5
  • TATA-box-binding protein
KeywordsPROTEIN TRANSPORT / nuclear transport receptor Kap114p / yeast TATA-box binding protein
Function / homology
Function and homology information


TFIIA-class transcription factor complex binding / RNA polymerase III transcription regulatory region sequence-specific DNA binding / RNA polymerase III preinitiation complex assembly / transcription factor TFIIIB complex / RNA polymerase I general transcription initiation factor binding / regulation of transcription by RNA polymerase III / RNA polymerase I preinitiation complex assembly / transcription factor TFIIA complex / NLS-bearing protein import into nucleus / DNA binding, bending ...TFIIA-class transcription factor complex binding / RNA polymerase III transcription regulatory region sequence-specific DNA binding / RNA polymerase III preinitiation complex assembly / transcription factor TFIIIB complex / RNA polymerase I general transcription initiation factor binding / regulation of transcription by RNA polymerase III / RNA polymerase I preinitiation complex assembly / transcription factor TFIIA complex / NLS-bearing protein import into nucleus / DNA binding, bending / nuclear import signal receptor activity / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA polymerase II transcribes snRNA genes / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / RNA Polymerase II Transcription Initiation / RNA Polymerase II Transcription Initiation And Promoter Clearance / RNA Polymerase II Pre-transcription Events / transcription factor TFIID complex / RNA polymerase II general transcription initiation factor activity / RNA Polymerase I Promoter Escape / nucleolar large rRNA transcription by RNA polymerase I / Estrogen-dependent gene expression / mRNA transport / nuclear pore / RNA polymerase II core promoter sequence-specific DNA binding / RNA polymerase II preinitiation complex assembly / DNA-templated transcription initiation / small GTPase binding / protein import into nucleus / disordered domain specific binding / nuclear envelope / DNA-binding transcription factor binding / transcription regulator complex / RNA polymerase II-specific DNA-binding transcription factor binding / negative regulation of DNA-templated transcription / chromatin binding / positive regulation of transcription by RNA polymerase II / protein-containing complex / nucleoplasm / nucleus / cytosol / cytoplasm
Similarity search - Function
Importin-beta N-terminal domain / Importin-beta N-terminal domain / Importin-beta N-terminal domain profile. / Importin-beta, N-terminal domain / TATA-box binding protein, eukaryotic / TATA-box binding protein / TATA-box binding protein, conserved site / Transcription factor TFIID (or TATA-binding protein, TBP) / Transcription factor TFIID repeat signature. / TBP domain superfamily ...Importin-beta N-terminal domain / Importin-beta N-terminal domain / Importin-beta N-terminal domain profile. / Importin-beta, N-terminal domain / TATA-box binding protein, eukaryotic / TATA-box binding protein / TATA-box binding protein, conserved site / Transcription factor TFIID (or TATA-binding protein, TBP) / Transcription factor TFIID repeat signature. / TBP domain superfamily / Armadillo-like helical / Armadillo-type fold
Similarity search - Domain/homology
TATA-box-binding protein / Importin subunit beta-5
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.03 Å
AuthorsHsia, K.C. / Liao, C.C. / Wang, C.H. / Wu, Y.M.
Funding support Taiwan, 1items
OrganizationGrant numberCountry
Academia Sinica (Taiwan) Taiwan
CitationJournal: Nat Commun / Year: 2023
Title: Structural convergence endows nuclear transport receptor Kap114p with a transcriptional repressor function toward TATA-binding protein.
Authors: Chung-Chi Liao / Yi-Sen Wang / Wen-Chieh Pi / Chun-Hsiung Wang / Yi-Min Wu / Wei-Yi Chen / Kuo-Chiang Hsia /
Abstract: The transcription factor TATA-box binding protein (TBP) modulates gene expression in nuclei. This process requires the involvement of nuclear transport receptors, collectively termed karyopherin-β ...The transcription factor TATA-box binding protein (TBP) modulates gene expression in nuclei. This process requires the involvement of nuclear transport receptors, collectively termed karyopherin-β (Kap-β) in yeast, and various regulatory factors. In previous studies we showed that Kap114p, a Kap-β that mediates nuclear import of yeast TBP (yTBP), modulates yTBP-dependent transcription. However, how Kap114p associates with yTBP to exert its multifaceted functions has remained elusive. Here, we employ single-particle cryo-electron microscopy to determine the structure of Kap114p in complex with the core domain of yTBP (yTBP). Remarkably, Kap114p wraps around the yTBP N-terminal lobe, revealing a structure resembling transcriptional regulators in complex with TBP, suggesting convergent evolution of the two protein groups for a common function. We further demonstrate that Kap114p sequesters yTBP away from promoters, preventing a collapse of yTBP dynamics required for yeast responses to environmental stress. Hence, we demonstrate that nuclear transport receptors represent critical elements of the transcriptional regulatory network.
History
DepositionOct 12, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 20, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Importin subunit beta-5
B: TATA-box-binding protein


Theoretical massNumber of molelcules
Total (without water)134,1402
Polymers134,1402
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Importin subunit beta-5 / 114 kDa karyopherin / Karyopherin subunit beta-5 / Karyopherin-114


Mass: 114019.695 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: KAP114, YGL241W, HRC1004 / Production host: Escherichia coli (E. coli) / References: UniProt: P53067
#2: Protein TATA-box-binding protein / TATA sequence-binding protein / TBP / TATA-binding factor / TATA-box factor / Transcription factor ...TATA sequence-binding protein / TBP / TATA-binding factor / TATA-box factor / Transcription factor D / Transcription initiation factor TFIID TBP subunit


Mass: 20120.754 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: SPT15, BTF1, TBP1, YER148W / Production host: Escherichia coli (E. coli) / References: UniProt: P13393

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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: nuclear transport receptor Kap114p complex with yeast TATA-box binding protein
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae S228 (yeast)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
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 / Nominal defocus max: 25000 nm / Nominal defocus min: 15000 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 4.03 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 212899 / Symmetry type: POINT

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