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  • Didemnin B and ternatin-4 differentially inhibit conformational changes in eEF1A required for aminoacyl-tRNA accommodation into mammalian ribosomes.

Didemnin B and ternatin-4 differentially inhibit conformational changes in eEF1A required for aminoacyl-tRNA accommodation into mammalian ribosomes.

eLife (2022-10-21)
Manuel F Juette, Jordan D Carelli, Emily J Rundlet, Alan Brown, Sichen Shao, Angelica Ferguson, Michael R Wasserman, Mikael Holm, Jack Taunton, Scott C Blanchard
要旨

Rapid and accurate mRNA translation requires efficient codon-dependent delivery of the correct aminoacyl-tRNA (aa-tRNA) to the ribosomal A site. In mammals, this fidelity-determining reaction is facilitated by the GTPase elongation factor-1 alpha (eEF1A), which escorts aa-tRNA as an eEF1A(GTP)-aa-tRNA ternary complex into the ribosome. The structurally unrelated cyclic peptides didemnin B and ternatin-4 bind to the eEF1A(GTP)-aa-tRNA ternary complex and inhibit translation but have different effects on protein synthesis in vitro and in vivo. Here, we employ single-molecule fluorescence imaging and cryogenic electron microscopy to determine how these natural products inhibit translational elongation on mammalian ribosomes. By binding to a common site on eEF1A, didemnin B and ternatin-4 trap eEF1A in an intermediate state of aa-tRNA selection, preventing eEF1A release and aa-tRNA accommodation on the ribosome. We also show that didemnin B and ternatin-4 exhibit distinct effects on the dynamics of aa-tRNA selection that inform on observed disparities in their inhibition efficacies and physiological impacts. These integrated findings underscore the value of dynamics measurements in assessing the mechanism of small-molecule inhibition and highlight potential of single-molecule methods to reveal how distinct natural products differentially impact the human translation mechanism.

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製品内容

Sigma-Aldrich
Didemnin B, ≥95% (HPLC)
Millipore
抗FLAG® M2抗体 アフィニティーゲル, purified immunoglobulin, buffered aqueous glycerol solution
Sigma-Aldrich
ピルビン酸キナーゼ ウサギ筋肉由来, Type III, lyophilized powder, 350-600 units/mg protein
Sigma-Aldrich
Anti-EF1α Antibody, clone CBP-KK1, clone CBP-KK1, Upstate®, from mouse