Structure of the hypusinylated eukaryotic translation factor eIF-5A bound to the ribosome

Abstract

During protein synthesis, ribosomes become stalled on polyproline-containing sequences, unless they are rescued in archaea and eukaryotes by the initiation factor 5A (a/eIF-5A) and in bacteria by the homologous protein EF-P. While a structure of EF-P bound to the 70S ribosome exists, structural insight into eIF-5A on the 80S ribosome has been lacking. Here we present a cryo-electron microscopy reconstruction of eIF-5A bound to the yeast 80S ribosome at 3.9 Å resolution. The structure reveals that the unique and functionally essential post-translational hypusine modification reaches toward the peptidyltransferase center of the ribosome, where the hypusine moiety contacts A76 of the CCA-end of the P-site tRNA. These findings would support a model whereby eIF-5A stimulates peptide bond formation on polyproline-stalled ribosomes by stabilizing and orienting the CCA-end of the P-tRNA, rather than by directly contributing to the catalysis.

Crystal Structure of Hypusine-Containing Translation Factor eIF5A Bound to a Rotated Eukaryotic Ribosome

“…S2). This conformation of hypusine is similar to the hypusine conformation in the cryo-EM structure of the 80S ribosome/eIF5A complex [8], although the κ-amino group is slightly shifted toward residue A2808 of the 25S rRNA in the cryo-EM structure (Fig. 1c and S2).…”

“…1a). Although our crystal structure lacks a P-site tRNA, we found that eIF5A occupies the same position as was observed in the 3.9 Å cryo-EM structure of the 80S ribosome complex with A- and P-site tRNAs [8] (Fig. 2a and S1).…”

“…S1). As in the cryo-EM structure of the 80S ribosome/eIF5A complex [8], the hypusine residue is located in direct vicinity of the ribosomal P site. The hypusine residue adopts a sharply bent conformation in which its positively charged κ-amino group is directed toward the CCA-end of the P-site tRNA, pointing between the 2′- and 3′-OH groups on the C75 ribose (Fig.…”

“…In eukaryotes, eIF5A activity depends on its unusual post-translationally modified residue hypusine, which is thought to augment the rate of peptide bond formation in the ribosomal catalytic center [4–6]. Structural studies of EF-P bound to the bacterial 70S ribosome from Thermus thermophilus and of eIF5A bound to the eukaryotic 80S ribosome from yeast showed that both EF-P and eIF5A bind the ribosome in the vicinity of the ribosomal catalytic center [1,7,8], positioning the critical hypusine residue (in the eIF5A structure) to contact the acceptor stem of the P-site tRNA [8]. Although these studies provided mechanistic insights into EF-P/eIF5A activity, in both structural studies the ribosome was in the same classical state conformation.…”

Polyamines in Eukaryotes, Bacteria, and Archaea

“…Firstly, the aminobutyl group of Spd is required for the hypusine posttranslational modification of translation factor eIF5A, which is required for the translation of mRNAs encoding polyproline tracts (1)(2)(3)(4)(5)(6). The enzyme deoxyhypusine synthase (DHS) transfers the aminobutyl group of Spd to eIF5A and is encoded in all eukaryotic genomes, including some intracellular parasites that have lost their polyamine biosynthetic pathway (7).…”