Conformational dynamics of eukaryotic translation.

Abstract

The ribosome is a molecular machine that adopts multiple conformations during translation. The body of the small ribosomal subunit rotates relatively to the large ribosomal subunit by about 9 degrees. This movement reconfigures tRNA binding sites, which results in a partial movement of tRNAs on the ribosome. At the beginning of the elongation cycle, the ribosome awaits A-site decoding and occupies the non-rotated conformation. Upon A-site tRNA binding, the ribosome rapidly catalyzes peptidyl transfer. It is followed by counterclockwise rotations of the small ribosomal subunit that puts the ribosome into the rotated state. The resultant pre-translocation complex is dynamic and exchanges between rotated and non-rotated conformations. Eukaryotic elongation factor 2 (eEF2) catalyzes translocation, which moves tRNA-mRNA and resets the ribosome into the non-rotated state. Despite extensive biochemical and structural studies, the underlying mechanism of translation elongation remains elusive. We developed a single-molecule fluorescence resonance energy transfer (smFRET) system that allows the observation of intersubunit conformational change of yeast ribosomes in real-time. We followed the ribosome conformational change in pre- and post-translocation ribosome complexes. To interrogate the mechanism of translocation, we applied antifungal sordarin. Sordarin stabilizes eEF2 on the ribosome after GTP hydrolysis and phosphate release. Our results suggest that in the presence of sordarin, the mid and late stages of translocation are thermally driven.