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TitleStructure of the translating ribosome arrested by cycloheximide.
Journal, issue, pagesProc Natl Acad Sci U S A, Vol. 118, Issue 48, Year 2021
Publish dateNov 30, 2021
AuthorsLunda Shen / Zhaoming Su / Kailu Yang / Cheng Wu / Thomas Becker / Deborah Bell-Pedersen / Junjie Zhang / Matthew S Sachs /
PubMed AbstractRibosomes translate RNA into proteins. The protein synthesis inhibitor cycloheximide (CHX) is widely used to inhibit eukaryotic ribosomes engaged in translation elongation. However, the lack of ...Ribosomes translate RNA into proteins. The protein synthesis inhibitor cycloheximide (CHX) is widely used to inhibit eukaryotic ribosomes engaged in translation elongation. However, the lack of structural data for actively translating polyribosomes stalled by CHX leaves unanswered the question of which elongation step is inhibited. We elucidated CHX's mechanism of action based on the cryo-electron microscopy structure of actively translating ribosomes bound with CHX at 2.7-Å resolution. The ribosome structure from this filamentous fungus contains clearly resolved ribosomal protein eL28, like higher eukaryotes but unlike budding yeast, which lacks eL28. Despite some differences in overall structures, the ribosomes from , yeast, and humans all contain a highly conserved CHX binding site. We also sequenced classic CHX-resistant alleles. These mutations, including one at a residue not previously observed to affect CHX resistance in eukaryotes, were in the large subunit proteins uL15 and eL42 that are part of the CHX-binding pocket. In addition to A-site transfer RNA (tRNA), P-site tRNA, messenger RNA, and CHX that are associated with the translating ribosome, spermidine is present near the CHX binding site close to the E site on the large subunit. The tRNAs in the peptidyl transferase center are in the A/A site and the P/P site. The nascent peptide is attached to the A-site tRNA and not to the P-site tRNA. The structural and functional data obtained show that CHX arrests the ribosome in the classical PRE translocation state and does not interfere with A-site reactivity.
External linksProc Natl Acad Sci U S A / PubMed:34815343 / PubMed Central
MethodsEM (single particle)
Resolution2.7 Å
Structure data

EMDB-24307, PDB-7r81:
Structure of the translating Neurospora crassa ribosome arrested by cycloheximide
Method: EM (single particle) / Resolution: 2.7 Å

Chemicals

ChemComp-3HE:
4-{(2R)-2-[(1S,3S,5S)-3,5-dimethyl-2-oxocyclohexyl]-2-hydroxyethyl}piperidine-2,6-dione / Cycloheximide

ChemComp-SPD:
SPERMIDINE / Spermidine

ChemComp-MG:
Unknown entry

Source
  • neurospora crassa (fungus)
KeywordsTRANSLATION / Protein Synthesis Inhibitor / Cytosolic Ribosome / Polysomal Ribosome / Peptidyl Transferase Center

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