TY - JOUR TI - Didemnin B and ternatin-4 differentially inhibit conformational changes in eEF1A required for aminoacyl-tRNA accommodation into mammalian ribosomes AU - Juette, Manuel F AU - Carelli, Jordan D AU - Rundlet, Emily J AU - Brown, Alan AU - Shao, Sichen AU - Ferguson, Angelica AU - Wasserman, Michael R AU - Holm, Mikael AU - Taunton, Jack AU - Blanchard, Scott C A2 - Hinnebusch, Alan G A2 - Manley, James L A2 - Aitken, Colin Echeverría A2 - Puglisi, Joseph D A2 - Walker, Sarah E VL - 11 PY - 2022 DA - 2022/10/20 SP - e81608 C1 - eLife 2022;11:e81608 DO - 10.7554/eLife.81608 UR - https://doi.org/10.7554/eLife.81608 AB - 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. KW - smFRET KW - Didemnin KW - ribosome KW - eEF1A KW - Ternatin KW - o. cuniculus JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -