Mutations at the accommodation gate of the ribosome impair RF2-dependent translation termination

Abstract

During protein synthesis, aminoacyl-tRNA (aa-tRNA) and release factors 1 and 2 (RF1 and RF2) have to bind at the catalytic center of the ribosome on the 50S subunit where they take part in peptide bond formation or peptidyl-tRNA hydrolysis, respectively. Computer simulations of aa-tRNA movement into the catalytic site (accommodation) suggested that three nucleotides of 23S rRNA, U2492, C2556, and C2573, form a ''gate'' at which aa-tRNA movement into the A site is retarded. Here we examined the role of nucleotides C2573 of 23S rRNA, a part of the putative accommodation gate, and of the neighboring A2572 for aa-tRNA binding followed by peptide bond formation and for the RF2-dependent peptide release. Mutations at the two positions did not affect aa-tRNA accommodation, peptide bond formation, or the fidelity of aa-tRNA selection, but impaired RF2-catalyzed peptide release. The data suggest that the ribosome is a robust machine that allows rapid aa-tRNA accommodation despite the defects at the accommodation gate. In comparison, peptide release by RF2 appears more sensitive to these mutations, due to slower accommodation of the factor or effects on RF2 positioning in the A site.

Mutations of highly conserved bases in the peptidyltransferase center induce compensatory rearrangements in yeast ribosomes

“…In addition, a recent study suggested that peptide bond formation and not accommodation may actually be the rate-limiting step in the translation elongation cycle (Johansson et al 2011). The observations that mutation of critical bases in the tRNA accommodation corridor did not affect accommodation rates (Burakovsky et al 2010), coupled with the observations in the present study, support this notion.…”

“…This suggests that rRNA in these functional regions of the ribosome formed compensatory rearrangements in response to single base substitutions at targeted positions. This further supports the idea that the ribosome is a robust machine (Burakovsky et al 2010) and can adjust its structure to preserve function.…”

“…NMR studies suggested that this nucleotide is conformationally flexible and is functionally involved in the mechanism of translation (Blanchard and Puglisi 2001). Similarly, the C2942U allele of the second gate position was the only viable mutation at this position in yeast, but all three possible base substitutions were viable and did not show significant growth phenotypes in bacteria (Burakovsky et al 2010). These observations suggest that, despite the high degree of evolutionary conservation of these nucleotides, certain processes are differentially fine-tuned in eukaryotic as compared to prokaryotic ribosomes.…”

“…In addition, mutation A2572U (yeast A2941U) promoted resistance to the PTC antibiotic valnemulin in Mycobacterium smegmatis (Long et al 2009). In E. coli, various mutations at positions A2572 and C2573 (yeast A2941 and C2942) did not affect aa-tRNA accommodation but promoted decreased rates of RF2-dependent peptidyltRNA hydrolysis (Burakovsky et al 2010). However, the three ''gate bases,'' as well as A2572 (yeast A2941), changed their conformations in response to different functionally important ribosomal mutations, suggesting that they are a part of the functional communication network within the ribosome (Beringer et al 2005;Meskauskas and Dinman 2007;Petrov et al 2008).…”

Evolutionary optimization of speed and accuracy of decoding on the ribosome

“…Indeed, mutagenesis studies [61] indicated that substitutions of residues U2492 and U2555 at the accommodation gate decreased the fidelity of translation, supporting the view that the accommodation gate may attenuate aa-tRNA binding. However, mutations of C2573 and the neighbouring A2572 did not affect aa-tRNA accommodation, peptide bond formation or the fidelity of aa-tRNA selection, suggesting that the ribosome may allow rapid aa-tRNA accommodation in spite of defects at the accommodation gate, perhaps by using a different pathway [62]. Alternatively, misalignment of the near-cognate tRNA may disturb the network of interactions between tRNA and ribosome [56], thereby disfavouring accommodation and favouring rejection.…”