SUMMARY Translation factor eIF5A, containing the unique amino acid hypusine, was originally shown to stimulate methionyl-puromycin synthesis, a model assay for peptide bond formation. More recently, eIF5A was shown to promote translation elongation; however, its precise requirement in protein synthesis has remained elusive. Here we use in vivo assays in yeast and in vitro reconstituted translation assays to reveal a specific requirement for eIF5A to promote peptide-bond formation between consecutive proline residues. Addition of eIF5A relieves ribosomal stalling during translation of three consecutive proline residues in vitro, and loss of eIF5A function impairs translation of polyproline-containing proteins in vivo. Hydroxyl radical probing experiments localized eIF5A near the E site of the ribosome with its hypusine residue adjacent to the acceptor stem of the P-site tRNA. Thus, eIF5A, like its bacterial ortholog EFP, is proposed to stimulate the peptidyl-transferase activity of the ribosome and facilitate the reactivity of poor substrates like proline.

Translation elongation factors facilitate protein synthesis by the ribosome. Previous studies identified two universally conserved translation elongation factors EF-Tu/eEF1A and EF-G/eEF2 that deliver aminoacyl-tRNAs to the ribosome and promote ribosomal translocation, respectively1. The factor eIF5A, the sole protein in eukaryotes and archaea containing the unusual amino acid hypusine [Nε-(4-amino-2-hydroxybutyl)lysine]2, was originally identified based on its ability to stimulate the yield (endpoint) of methionyl-puromycin synthesis, a model assay for first peptide bond synthesis thought to report on certain aspects of translation initiation3,4. Hypusine is required for eIF5A to associate with ribosomes5,6, and to stimulate methionyl-puromycin synthesis7. As eIF5A did not stimulate earlier steps of translation initiation8, and depletion of eIF5A in yeast only modestly impaired protein synthesis9, it was proposed that eIF5A function was limited to stimulating synthesis of the first peptide bond or that eIF5A functioned on only a subset of cellular mRNAs. However, the precise cellular role of eIF5A is unknown, and the protein has also been linked to mRNA decay, including the nonsense-mediated mRNA decay (NMD) pathway10,11, and to nucleocytoplasmic transport12,13. Here we show using molecular genetic and biochemical studies that eIF5A promotes translation elongation. Depletion or inactivation of eIF5A in yeast resulted in the accumulation of polysomes and an increase in ribosomal transit times. Addition of recombinant eIF5A from yeast, but not a derivative lacking hypusine, enhanced the rate of tripeptide synthesis in vitro. Moreover, inactivation of eIF5A mimicked the effects of the eEF2 inhibitor sordarin, indicating that eIF5A might function together with eEF2 to promote ribosomal translocation. As eIF5A is a structural homolog of the bacterial protein EF-P14,15, we propose that eIF5A/EF-P is a universally conserved translation elongation factor.