negative regulation of glucose mediated signaling pathway / negative regulation of translational frameshifting / mTORC1-mediated signalling / Protein hydroxylation / ribosome-associated ubiquitin-dependent protein catabolic process / GDP-dissociation inhibitor activity / pre-mRNA 5'-splice site binding / Formation of the ternary complex, and subsequently, the 43S complex / Translation initiation complex formation / nonfunctional rRNA decay ...negative regulation of glucose mediated signaling pathway / negative regulation of translational frameshifting / mTORC1-mediated signalling / Protein hydroxylation / ribosome-associated ubiquitin-dependent protein catabolic process / GDP-dissociation inhibitor activity / pre-mRNA 5'-splice site binding / Formation of the ternary complex, and subsequently, the 43S complex / Translation initiation complex formation / nonfunctional rRNA decay / Ribosomal scanning and start codon recognition / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / response to cycloheximide / Major pathway of rRNA processing in the nucleolus and cytosol / mRNA destabilization / SRP-dependent cotranslational protein targeting to membrane / GTP hydrolysis and joining of the 60S ribosomal subunit / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / Formation of a pool of free 40S subunits / negative regulation of mRNA splicing, via spliceosome / preribosome, large subunit precursor / L13a-mediated translational silencing of Ceruloplasmin expression / regulation of amino acid metabolic process / G-protein alpha-subunit binding / positive regulation of protein kinase activity / 90S preribosome / endonucleolytic cleavage to generate mature 3'-end of SSU-rRNA from (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / regulation of translational fidelity / protein-RNA complex assembly / ribosomal subunit export from nucleus / translation regulator activity / endonucleolytic cleavage in ITS1 to separate SSU-rRNA from 5.8S rRNA and LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of LSU-rRNA / cellular response to amino acid starvation / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / rescue of stalled ribosome / ribosomal large subunit biogenesis / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of SSU-rRNA / small-subunit processome / translational initiation / positive regulation of apoptotic signaling pathway / macroautophagy / protein kinase C binding / maintenance of translational fidelity / cytoplasmic stress granule / rRNA processing / ribosome biogenesis / ribosome binding / ribosomal small subunit biogenesis / ribosomal small subunit assembly / small ribosomal subunit / small ribosomal subunit rRNA binding / 5S rRNA binding / ribosomal large subunit assembly / large ribosomal subunit rRNA binding / cytosolic small ribosomal subunit / cytosolic large ribosomal subunit / cytoplasmic translation / negative regulation of translation / rRNA binding / ribosome / structural constituent of ribosome / translation / G protein-coupled receptor signaling pathway / negative regulation of gene expression / response to antibiotic / mRNA binding / nucleolus / mitochondrion / RNA binding / zinc ion binding / nucleoplasm / nucleus / metal ion binding / cytosol / cytoplasm Similarity search - Function
: / : / : / Ribosomal protein S26e signature. / Ribosomal protein L41 / Ribosomal protein L41 / Ribosomal protein S21e, conserved site / Ribosomal protein S21e signature. / Ribosomal protein S26e / Ribosomal protein S26e superfamily ...: / : / : / Ribosomal protein S26e signature. / Ribosomal protein L41 / Ribosomal protein L41 / Ribosomal protein S21e, conserved site / Ribosomal protein S21e signature. / Ribosomal protein S26e / Ribosomal protein S26e superfamily / Ribosomal protein S26e / Ribosomal protein L29e / Ribosomal L29e protein family / Ribosomal protein S19e, conserved site / Ribosomal protein S19e signature. / Ribosomal protein L13e, conserved site / Ribosomal protein L13e signature. / Ribosomal protein S5, eukaryotic/archaeal / Ribosomal protein S21e / Ribosomal protein S21e superfamily / Ribosomal protein S21e / Ribosomal protein S2, eukaryotic / Small (40S) ribosomal subunit Asc1/RACK1 / Ribosomal protein L22e / Ribosomal protein L22e superfamily / Ribosomal L22e protein family / Ribosomal protein L38e / Ribosomal protein L38e superfamily / Ribosomal L38e protein family / 40S Ribosomal protein S10 / Ribosomal protein L27e, conserved site / Ribosomal protein L27e signature. / Ribosomal protein L10e, conserved site / Ribosomal protein L44e signature. / Ribosomal protein L10e signature. / Ribosomal protein L10e / Plectin/S10, N-terminal / Ribosomal protein L19, eukaryotic / Plectin/S10 domain / Ribosomal protein L13e / Ribosomal protein L13e / Ribosomal protein S10, eukaryotic/archaeal / 60S ribosomal protein L18a/ L20, eukaryotes / Ribosomal protein S8e subdomain, eukaryotes / Ribosomal protein L19/L19e conserved site / : / Ribosomal protein L19e signature. / : / Ribosomal protein L24e, conserved site / Ribosomal protein S7e signature. / Ribosomal protein L24e signature. / Ribosomal protein S25 / S25 ribosomal protein / Ribosomal protein L44e / Ribosomal protein L44 / : / Ribosomal protein L34e, conserved site / Ribosomal protein L34e signature. / Ribosomal protein L5 eukaryotic, C-terminal / Ribosomal L18 C-terminal region / Ribosomal protein L6e signature. / Ribosomal protein S17e, conserved site / Ribosomal protein L30e signature 1. / Ribosomal protein S17e signature. / Ribosomal protein S2, eukaryotic/archaeal / 50S ribosomal protein L18Ae/60S ribosomal protein L20 and L18a / Ribosomal protein L23/L25, N-terminal / Ribosomal protein 50S-L18Ae/60S-L20/60S-L18A / Ribosomal proteins 50S-L18Ae/60S-L20/60S-L18A / Ribosomal protein L23, N-terminal domain / Ribosomal protein S3Ae, conserved site / Ribosomal protein S3Ae signature. / 40S ribosomal protein S29/30S ribosomal protein S14 type Z / Eukaryotic Ribosomal Protein L27, KOW domain / 40S ribosomal protein S4, C-terminal domain / 40S ribosomal protein S4 C-terminus / Ribosomal protein S4e, N-terminal, conserved site / Ribosomal protein S4e signature. / Ribosomal protein 60S L18 and 50S L18e / Ribosomal protein S3, eukaryotic/archaeal / Ribosomal protein L30e signature 2. / Ribosomal protein L27e / Ribosomal protein L18/L18-A/B/e, conserved site / Ribosomal protein L27e superfamily / Ribosomal L27e protein family / Ribosomal protein L18e signature. / Ribosomal protein L36e signature. / Ribosomal protein L30e, conserved site / Ribosomal protein S27e signature. / Ribosomal protein S19e / Ribosomal protein S19e / Ribosomal_S19e / Ribosomal protein L39e, conserved site / Ribosomal protein L39e signature. / Ribosomal protein L35Ae, conserved site / Ribosomal protein L35Ae signature. / Ribosomal protein L34Ae / Ribosomal protein L34e / Ribosomal protein S8e, conserved site / Ribosomal protein S8e signature. Similarity search - Domain/homology
Rps5p / Small ribosomal subunit protein eS1 / RPS22A isoform 1 / RPL38 isoform 1 / RPL10 isoform 1 / RPS29A isoform 1 / RPS20 isoform 1 / RPS2 isoform 1 / 60S ribosomal protein L29 / 60S ribosomal protein L8 ...Rps5p / Small ribosomal subunit protein eS1 / RPS22A isoform 1 / RPL38 isoform 1 / RPL10 isoform 1 / RPS29A isoform 1 / RPS20 isoform 1 / RPS2 isoform 1 / 60S ribosomal protein L29 / 60S ribosomal protein L8 / RPL41A isoform 1 / RPL24A isoform 1 / RPL11B isoform 1 / 40S ribosomal protein S25 / RPL9A isoform 1 / 40S ribosomal protein S26 / RPL5 isoform 1 / 40S ribosomal protein S8 / RPL32 isoform 1 / 40S ribosomal protein S3 / RPL4A isoform 1 / Large ribosomal subunit protein uL3 / RPS15 isoform 1 / RPS28A isoform 1 / Small ribosomal subunit protein uS4A / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL39 / Large ribosomal subunit protein uL30A / Large ribosomal subunit protein uL22A / Large ribosomal subunit protein uL24A / Large ribosomal subunit protein eL33A / Large ribosomal subunit protein eL36A / Large ribosomal subunit protein eL15A / Large ribosomal subunit protein eL22A / Small ribosomal subunit protein uS15 / Small ribosomal subunit protein eS19A / Small ribosomal subunit protein eS21A / Large ribosomal subunit protein eL27A / Large ribosomal subunit protein eL31A / Large ribosomal subunit protein eL20A / Large ribosomal subunit protein eL43A / Large ribosomal subunit protein eL42A / Small ribosomal subunit protein uS12A / Small ribosomal subunit protein eS24A / Small ribosomal subunit protein eS4A / Small ribosomal subunit protein eS6A / Large ribosomal subunit protein uL14A / Large ribosomal subunit protein uL2A / Small ribosomal subunit protein uS17A / Large ribosomal subunit protein eL18A / Small ribosomal subunit protein uS9A / Small ribosomal subunit protein uS13A / Large ribosomal subunit protein eL19A / Large ribosomal subunit protein uL29A / Large ribosomal subunit protein eL30 / Small ribosomal subunit protein eS17B / Large ribosomal subunit protein uL13A / Small ribosomal subunit protein eS7A / Small ribosomal subunit protein uS2A / Small ribosomal subunit protein eS27A / Large ribosomal subunit protein eL14A / Small ribosomal subunit protein RACK1 / Small ribosomal subunit protein uS11B / Large ribosomal subunit protein eL37A / Large ribosomal subunit protein eL34A / Large ribosomal subunit protein eL6A / Large ribosomal subunit protein eL21A / Small ribosomal subunit protein eS10A / Large ribosomal subunit protein eL13A Similarity search - Component
Biological species
Saccharomyces cerevisiae (brewer's yeast)
Method
single particle reconstruction / cryo EM / Resolution: 3.2 Å
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)
5R00ES025835-05
United States
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2020 Title: Structural impact of K63 ubiquitin on yeast translocating ribosomes under oxidative stress. Authors: Ye Zhou / Panagiotis L Kastritis / Shannon E Dougherty / Jonathan Bouvette / Allen L Hsu / Laura Burbaum / Shyamal Mosalaganti / Stefan Pfeffer / Wim J H Hagen / Friedrich Förster / Mario J ...Authors: Ye Zhou / Panagiotis L Kastritis / Shannon E Dougherty / Jonathan Bouvette / Allen L Hsu / Laura Burbaum / Shyamal Mosalaganti / Stefan Pfeffer / Wim J H Hagen / Friedrich Förster / Mario J Borgnia / Christine Vogel / Martin Beck / Alberto Bartesaghi / Gustavo M Silva / Abstract: Subpopulations of ribosomes are responsible for fine tuning the control of protein synthesis in dynamic environments. K63 ubiquitination of ribosomes has emerged as a new posttranslational ...Subpopulations of ribosomes are responsible for fine tuning the control of protein synthesis in dynamic environments. K63 ubiquitination of ribosomes has emerged as a new posttranslational modification that regulates protein synthesis during cellular response to oxidative stress. K63 ubiquitin, a type of ubiquitin chain that functions independently of the proteasome, modifies several sites at the surface of the ribosome, however, we lack a molecular understanding on how this modification affects ribosome structure and function. Using cryoelectron microscopy (cryo-EM), we resolved the first three-dimensional (3D) structures of K63 ubiquitinated ribosomes from oxidatively stressed yeast cells at 3.5-3.2 Å resolution. We found that K63 ubiquitinated ribosomes are also present in a polysome arrangement, similar to that observed in yeast polysomes, which we determined using cryoelectron tomography (cryo-ET). We further showed that K63 ubiquitinated ribosomes are captured uniquely at the rotated pretranslocation stage of translation elongation. In contrast, cryo-EM structures of ribosomes from mutant cells lacking K63 ubiquitin resolved at 4.4-2.7 Å showed 80S ribosomes represented in multiple states of translation, suggesting that K63 ubiquitin regulates protein synthesis at a selective stage of elongation. Among the observed structural changes, ubiquitin mediates the destabilization of proteins in the 60S P-stalk and in the 40S beak, two binding regions of the eukaryotic elongation factor eEF2. These changes would impact eEF2 function, thus, inhibiting translocation. Our findings help uncover the molecular effects of K63 ubiquitination on ribosomes, providing a model of translation control during oxidative stress, which supports elongation halt at pretranslocation.
History
Deposition
Jun 21, 2020
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Header (metadata) release
Aug 26, 2020
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Map release
Aug 26, 2020
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Update
Oct 23, 2024
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Current status
Oct 23, 2024
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Name: ZINC ION / type: ligand / ID: 76 / Number of copies: 6 / Formula: ZN
Molecular weight
Theoretical: 65.409 Da
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
-
Sample preparation
Buffer
pH: 7.5
Grid
Model: Quantifoil R2/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY
Vitrification
Cryogen name: ETHANE
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Frames/image: 1-7 / Number real images: 5243 / Average electron dose: 25.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
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