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. 2016 Feb 29;44(4):1944-51.
doi: 10.1093/nar/gkv1517. Epub 2015 Dec 28.

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

Christian Schmidt  1 , Thomas Becker  1 , André Heuer  1 , Katharina Braunger  1 , Vivekanandan Shanmuganathan  1 , Markus Pech  1 , Otto Berninghausen  1 , Daniel N Wilson  2 , Roland Beckmann  3
Affiliations

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

Christian Schmidt et al. Nucleic Acids Res. .

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.

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Figures

Figure 1.
Figure 1.
Cryo-EM structure of eIF-5A bound to the yeast 80S ribosome. (A) Transverse section of the cryo-EM map of eIF-5A–80S complex, (40S, yellow; 60S, gray), revealing the binding site of eIF-5A (dark red), P-tRNA (green) and A-tRNA (blue). (B) Comparison of ribosome binding positions of eIF-5A, EF-P (16) and E-site tRNA(48), relative to A-tRNA (blue) and P-tRNA (green). The domains for eIF-5A and EF-P are colored according to (C). (C) Schematic representation of the domain structures of eIF-5A, aIF-5A and EF-P. (D) Molecular model for the interaction of domains I (DI, red) and II (DII, orange) as well as N-terminal extension (NTE, magenta) of eIF-5A with rRNA and ribosomal protein components of the ribosome (gray). Ribosomal insert shows the orientation of the view.
Figure 2.
Figure 2.
Interaction of eIF-5A with the yeast 80S ribosome. (A) View of the NTE of eIF-5A sandwiched between ribosomal proteins uL1 (blue) and eL42 (tan). (B) Domain II (DII, orange) of eIF-5A inserts into the cleft between domains 1 and 2 of uL1 (blue). (C) Comparison of the ribosome binding position of domain I of eIF-5A (red), P-tRNA (green) and 25S rRNA helix 74 (H74, blue) relative to the EF-P, P-tRNA and H74 (gray) from the bacterial EF-P–70S complex (16). Arg27 of eIF-5A makes a potential hydrogen bond interaction (dashed line) with the P-tRNA (green). (D) Possible hydrogen bond interactions (dashed lines) between domain I of eIF-5A (red) with 25S rRNA nucleotides within helices H74, H80 and H93 of the ribosome (gray).
Figure 3.
Figure 3.
Hypusine of eIF-5A at the peptidyltransferase center of the ribosome. (A) Electron density (gray mesh) and molecular model (red) for hypusine 51 (Hyp51) of eIF-5A. (B) Potential hydrogen bond interactions (dashed lines) between hypusine 51 (Hyp51) of eIF-5A (red) with the CCA-end of the P-tRNA (green) and 25S rRNA nucleotides within helix H74 (gray). (C) Electron density (gray mesh) and molecular models for the CCA-ends of P-tRNA (green) and A-tRNA with nascent chain (NC) (blue) as well as eIF-5A (red). (D) Comparison of A-tRNA (blue) and P-tRNA (green) from eIF-5A–80S complex with A- and P-tRNAs from post-attack complex (33) (gray). (E) Potential hydrogen bond interactions (dashed lines) between the loop of uL16 (orange), A-tRNA (blue) and P-tRNA (green). (F) Proximity of Asp108 of uL16 (orange) to the 3′ OH of A76 of the P-tRNA (8.9 Å) in the eIF-5A–80S complex, compared with the proximity of Ala2 of L27 (gray) to the 3′ OH of A76 of the P-tRNA (7.1 Å) in the pre-attack complex (33). The CCA-end of the A-tRNA (blue) and hypusine 51 (Hyp51) of eIF-5A (red) are shown for reference.
Figure 4.
Figure 4.
Model for eIF-5A action on the ribosome. (A) Certain nascent polypeptide chains, such as those containing polyproline stretches, destabilize the P-tRNA (green) and prevent peptide-bond formation with the incoming A-tRNA (blue). (B) The stalled ribosomes are recognized by eIF-5A, which binds such that the modified hypusine 51 (Hyp51) residue interacts with the A76 of the CCA-end of the P-tRNA. This interaction stabilizes the P-tRNA in the optimal geometry for peptide bond formation, leading to ordering of the loop of uL16, which in turn establishes interactions with both A- and P-tRNAs, facilitating efficient peptide bond formation. (C) Peptide bond formation leads to a deacylated tRNA in the P-site and A-tRNA bearing the nascent polypeptide chain extended by one amino acid.
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References

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