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.