Abstract
Translation is accompanied by rotation of the small and large ribosomal subunits relative to each other. Here, we use single-molecule Förster resonance energy transfer between fluorophores introduced into ribosomal proteins uS15 and eL30 to follow intersubunit dynamics of Saccharomyces cerevisiae ribosomes. Similar to their bacterial counterparts, yeast ribosomes are observed to sample two predominant FRET states corresponding to the nonrotated (NR) and rotated (R) conformations. Our data yield further evidence that intersubunit rotation is coupled to tRNA transitions between classical and hybrid binding states. The elongation cycle, which comprises tRNA binding, peptide transfer, and mRNA-tRNA translocation, is accompanied by switching from NR to R and then back to the NR conformation. We find that during mRNA-tRNA translocation mediated by the joint action of fungal elongation factor 3 (eEF3) and universally conserved elongation factor 2 (eEF2), eEF3 facilitates the late step of translocation, which follows eEF2-induced reverse intersubunit rotation. Our uS15-eL30 smFRET assay provides the basis for investigating eukaryotic mechanisms of translation regulation including ribosome pausing, stalling, and frameshifting.