The structure of the translated Neurospora ribosome captured by cycloheximide NASA

2021-11-24 01:55:48 By : Ms. Kitty Ke

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Edited by Joseph D. Puglisi, Stanford University School of Medicine, Stanford University, California, approved on October 26, 2021 (review received on June 28, 2021)

Ribosomes play a central role in the translation of RNA. The extended function of most eukaryotic ribosomes is blocked by the drug cycloheximide (CHX). The CHX binding site is known. In this work, we determined the structure of the translated ribosomes of filamentous fungi captured by CHX. By doing so, the specific state of the ribosome that is stalled in translation is revealed. This work is important because it provides a structural basis for the establishment of CHX's mechanism of action. It provides a model for fungal ribosomes, and compares its characteristics with those of other species to gain insight into the structure and function of eukaryotic ribosomes.

The ribosome translates RNA into protein. The protein synthesis inhibitor cycloheximide (CHX) is widely used to inhibit eukaryotic ribosomes involved in translation extension. However, due to the lack of structural data for active translation of polysomes blocked by CHX, the question of which extension step is inhibited cannot be answered. We clarified the mechanism of CHX based on the cryo-electron microscope structure, which actively translates the ribosome of Neurospora crassa with a resolution of 2.7 Å to bind to CHX. The ribosomal structure of this filamentous fungus contains the clearly resolved ribosomal protein eL28, which is similar to higher eukaryotes, but is different from budding yeast lacking eL28. Although there are some differences in the overall structure, ribosomes from Neurospora, yeast, and humans all contain highly conserved CHX binding sites. We also sequenced the classic Neurospora CHX resistance allele. These mutations, including those at residues that have not previously been observed to affect CHX resistance in eukaryotes, are located in the large subunit proteins uL15 and eL42 that are part of the CHX binding pocket. In addition to the A-site transfer RNA (tRNA), P-site tRNA, messenger RNA, and CHX associated with the ribosome translation of Neurospora crassa, spermidine also exists near the CHX binding site near the E site of the large subunit The tRNA in the center of the peptidyl transferase is located at the A/A site and the P/P site. The nascent peptide is linked to the A-site tRNA instead of the P-site tRNA. The obtained structural and functional data indicate that CHX prevents the ribosome in the classic PRE translocation state and does not interfere with the A site reaction.

↵1 Current address: State Key Laboratory of Biotherapy and Cancer Center, National Geriatrics Clinical Research Center, Department of Geriatrics, West China Hospital, Sichuan University, Chengdu 610044, Sichuan.

↵2 Current address: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford University, California 94305.

Author contributions: LS, ZS, KY, CW, TB, DB-P., JZ and MSS design research; LS, ZS, KY, CW and MSS conducted research; LS, ZS, KY, CW, TB, DB- P., JZ and MSS analyzed data; LS, ZS, KY, CW, TB, DB-P., JZ and MSS wrote this paper.

The author declares no competing interests.

This article is directly contributed by PNAS.

This article contains online support information https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2111862118/-/DCSupplemental.

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