Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation

Abstract

An 11.7-Å-resolution cryo-EM map of the yeast 80S . eEF2 complex in the presence of the antibiotic sordarin was interpreted in molecular terms, revealing large conformational changes within eEF2 and the 80S ribosome, including a rearrangement of the functionally important ribosomal intersubunit bridges. Sordarin positions domain III of eEF2 so that it can interact with the sarcinricin loop of 25S rRNA and protein rpS23 (S12p). This particular conformation explains the inhibitory action of sordarin and suggests that eEF2 is stalled on the 80S ribosome in a conformation that has similarities with the GTPase activation state. A ratchet-like subunit rearrangement (RSR) occurs in the 80S . eEF2 . sordarin complex that, in contrast to Escherichia coli 70S ribosomes, is also present in vacant 80S ribosomes. A model is suggested, according to which the RSR is part of a mechanism for moving the tRNAs during the translocation reaction.

Yeast Ribosomal/Cytochrome c SET Domain Methyltransferase Subfamily

“…However, it is clear that the interactions of methylated lysine residues in Rpl23a occur with RNA rather than with proteins. A similar situation is found for the methylated lysine residues of Rpl12ab (25,35,37). Thus, it appears that the interaction of methylated lysine residues in these ribosomal proteins with RNA is clearly different from the interaction of methylated lysine residues in histones with proteins (16, 18, 19, 33, 34).…”

“…These results suggest that methylation may function to correctly position the Rpl23ab/L14 protein within the large subunit. Interestingly, the position of Rpl23ab and its bridges to the small subunit RNA change during the translation cycle (37,38). Further studies will be needed to assay the effect of methylation on translational efficiency.…”

“…Structural reconstruction studies of the S. cerevisiae 80 S ribosome (35,37) and the Escherichia coli 70 S ribosome (38) have positioned the methylated residues of Rpl23ab and the corresponding region of the prokaryotic L14 homolog making contacts with the yeast 25 S or bacterial 23 S large subunit ribosomal RNA. These contacts would be expected to shield the methylated residues from interactions with other ribosomal proteins or cytosolic factors.…”

Sordarin Derivatives Induce a Novel Conformation of the Yeast Ribosome Translocation Factor eEF2

“…Advanced kinetic studies of the eukaryotic system are currently lacking, but the fundamental mechanism is believed to be conserved between the kingdoms, and both the ribosome and the elongation factor have high structural homology. This has been confirmed by cryo-EM 3 reconstructions of eEF2 in a sordarin-stabilized complex with the 80 S ribosome (8,9), which is rather similar to the 70 S-EF-G complex stabilized by fusidic acid (10). Both antibiotics prevent the release of the elongation factor from the ribosome after translocation (11).…”

“…Both antibiotics prevent the release of the elongation factor from the ribosome after translocation (11). In both cases domain IV of eEF2/EF-G protrudes into the A-site of the small ribosomal subunit, and the location of the remaining domains is also quite similar for eEF2 and EF-G (9,10). The function of EF-G/eEF2 appears to be dual, prior to translocation it promotes conformational changes within the pretranslocational ribosome-tRNA-mRNA complex, and after translocation it prevents peptidyl-tRNA from slipping back from the P-site.…”