eEF3 promotes late stages of tRNA translocation on the ribosome

In addition to the conserved translation elongation factors eEF1A and eEF2, fungi require a third essential elongation factor, eEF3. While eEF3 has been implicated in tRNA binding and release at the A and E sites, its exact mechanism of action is unclear. Here we show that eEF3 acts at the mRNA−tRNA translocation step by promoting the dissociation of the tRNA from the E site, but independent of aminoacyl−tRNA recruitment to the A site. Depletion of eEF3 in vivo leads to a general slow−down in translation elongation due to accumulation of ribosomes with an occupied A site. Cryo-EM analysis of ex vivo eEF3−ribosome complexes shows that eEF3 facilitates late steps of translocation by favoring non-rotated ribosomal states as well as by opening the L1 stalk to release the E-site tRNA. Additionally, our analysis provides structural insights into novel translation elongation states, enabling presentation of a revised yeast translation elongation cycle.