| dc.description.abstract | 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 microscopic structure of actively translating Neurospora crassa 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 Neurospora, yeast, and humans all contain a highly conserved CHX-binding site. We also sequenced classic Neurospora CHX-resistant alleles. These mutations, including one at a residue not previously observed to affect CHX-resistance in eukaryotes, were in large subunit proteins uL15 and eL42 that are part of the CHX binding pocket. In addition to A-site tRNA, P-site tRNA, mRNA, and CHX that are associated with the translating N. crassa ribosome, spermidine (SPD) is also 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 P/P site. The nascent peptide is attached to the A-site tRNA and not the P-site tRNA. The structural and functional data obtained show that CHX arrests the ribosome in the classical PRE state and does not interfere with A-site reactivity. | |
