Observing intersubunit dynamics in single yeast ribosomes

Translation is accompanied by the 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 the 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 the classical and hybrid binding states. In particular, the transition from the NR to R conformation is inhibited by the antibiotic cycloheximide, which is known to bind to the E site of the large subunit and prevent the movement of deacylated tRNA into the hybrid P/E state. 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 fungal elongation factor 3 (eEF3) stabilizes the NR conformation of the ribosome. Our data are consistent with the model suggesting that eEF3 facilitates E-site tRNA release at the late step of mRNA-tRNA translocation, following the reverse intersubunit rotation induced by the universally conserved elongation factor 2 (eEF2). Our uS15-eL30 smFRET assay provides the basis for investigating eukaryotic mechanisms of translation regulation, including ribosome pausing, stalling, and frameshifting.