ArticleMethods in enzymology2025
An smFRET assay to probe the impact of antibiotics on intersubunit rotation in eukaryotic ribosomes.
Article in Methods in enzymology, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Protein translation involves large-scale structural rearrangements within the ribosome. During translation elongation, the two subunits of the ribosome rotate with respect to each other to allow for tRNA-mRNA translocation. While conformational changes of bacterial ribosomes have been extensively explored using cryo-EM and single-molecule microscopy, structural dynamics of eukaryotic ribosomes are less well studied. Here we describe procedures for fluorescent labeling, assembly of elongation complexes and real-time observation of intersubunit rotation in yeast ribosomes using single-molecule Förster resonance energy transfer. We demonstrate that this assay can be used to interrogate mechanisms by which translation inhibitors perturb protein synthesis and alter ribosome structural dynamics.
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