Entropic Contribution of Elongation Factor P to Proline Positioning at the Catalytic Center of the Ribosome

Abstract

The peptide bond formation with the amino acid proline (Pro) on the ribosome is slow, resulting in translational stalling when several Pro have to be incorporated into the peptide. Stalling at poly-Pro motifs is alleviated by the elongation factor P (EF-P). Here we investigate why Pro is a poor substrate and how EF-P catalyzes the reaction. Linear free energy relationships of the reaction on the ribosome and in solution using 12 different Pro analogues suggest that the positioning of Pro-tRNA in the peptidyl transferase center is the major determinant for the slow reaction. With any Pro analogue tested, EF-P decreases the activation energy of the reaction by an almost uniform value of 2.5 kcal/mol. The main source of catalysis is the favorable entropy change brought about by EF-P. Thus, EF-P acts by entropic steering of Pro-tRNA toward a catalytically productive orientation in the peptidyl transferase center of the ribosome.

eIF5A facilitates translation termination globally and promotes the elongation of many non polyproline-specific tripeptide sequences

“…Our results are in line with previous studies which have described that most of these amino acids act badly during peptide bond formation, e.g. presence of a secondary amino group in proline has been associated with slow peptide bond formation and translation (1–6,27). Previous studies have also detected poor reactivity for glycine and for basic amino acids arginine and lysine (4,58).…”

“…Our in vivo study cannot rule out that the observed ribosome stalls could be provoked by the down regulation of other translation elongation factors. However, the direct binding of eIF5A to the 80S ribosome, and a role in peptide bond formation demonstrated by previous crystallography and biochemical studies (9,24–27), suggest that the effects we describe herein are also direct. The frequency of specific amino acids at the P- and A-sites in the eIF5A-regulated motifs suggests that eIF5A facilitates the formation of peptide bonds in situations of slow reactivity when glycine, proline, or an acidic amino acid, acts as a donor and when proline, glycine or a basic amino acid acts as an acceptor (Figure 6C).…”

“…Current data suggest that eIF5A could bind stalled ribosomes with a free E-site, and then eIF5A and its hypusine moiety would force a productive positioning of the CCA-end of the P-tRNA to facilitate the transfer of the nascent chain from P-tRNA to A-tRNA (26). Moreover, studies with EF-P have shown that the elongation factor contributes to the formation of a peptide bond with proline in an entropic way by providing a better orientation of Pro-tRNA for the catalytic reaction at the peptidyl transfer center (27). Therefore, these results explain how eIF5A promotes the elongation of paused ribosomes at polyproline motifs.…”

Resolving the α-glycosidic linkage of arginine-rhamnosylated translation elongation factor P triggers generation of the first ArgRha specific antibody

“…14–16 When EF-P is bound to the ribosome the rhamnosylated arginine protrudes towards the peptidyltransferase center thereby contributing to the favorable positioning of the peptidyl-Pro-tRNA and stabilization of the CCA-end of the prolyl-tRNA. 14,17,18 Loss of the rhamnose modification abolishes the pathogenicity of P. aeruginosa 14 and increases its susceptibility to certain antibiotics. 15 Thus inhibition of EF-P rhamnosylation might be a novel strategy to selectively suppress virulence.…”

Translation Control of Swarming Proficiency in Bacillus subtilis by 5-Amino-pentanolylated Elongation Factor P

“…remedies proline-induced pausing by binding to the ribosome near the pepitdyl transfer site and makes synthesis of oligoprolines entropically favorable (5). The presence of EF-P alone is not sufficient to maintain efficient translation of oligoprolines because the active protein also requires posttranslational modification (6 -9).…”