We study the mechanism and regulation of protein synthesis in eukaryotic cells focusing on regulation by GTP-binding (G) proteins and protein phosphorylation. The first step of protein synthesis is binding the initiator Met-tRNA to the small ribosomal subunit by the factor eIF2. The eIF2 is a GTP-binding protein and during the course of translation initiation the GTP is hydrolyzed to GDP. The eIF2 is released from the ribosome in complex with GDP and requires the guanine-nucleotide exchange factor eIF2B to convert eIF2-GDP to eIF2-GTP. This exchange reaction is regulated by a family of kinases that specifically phosphorylate the alpha subunit of eIF2 on serine at residue 51, and thereby covert eIF2 into an inhibitor of eIF2B. Among the family of eIF2alpha kinases are GCN2 (activated under conditions of amino acid starvation), PKR (activated by double-stranded RNA and downregulates protein synthesis in virally infected cells), and PERK (activated under conditions of ER stress). Our previous crystallography and accompanying molecular genetic analyses revealed that PKR helix alphaG contacts eIF2alpha on a face remote from the Ser51 phosphorylation site;however, the helix alphaG contact is critical for eIF2alpha phosphorylation. Moreover, when the structure of free eIF2alpha, in which the position of Ser51 is resolved, was docked on the structure of the PKReIF2alpha complex, Ser51 was 20 angstroms from the kinase active site. Mutation of Thr487 in PKR helix alphaG cripples the kinase and prevents eIF2alpha phosphorylation. To gain new insights into how Ser51 gains access to the PKR active site, we identified mutations in eIF2alpha that restored Ser51 phosphorylation both in vivo and in vitro. These eIF2alpha mutations disrupt a hydrophobic network that restricts the position of Ser51 and enhance the mobility of the loop containing Ser51. Moreover, binding of PKR to eIF2alpha enhanced the protease sensitivity of the Ser51 loop. Based on these studies and the structure of the PKReIF2alpha complex, we propose that docking of eIF2alpha on PKR induces a conformational change, greater than the spontaneous breathing of the Ser51 loop, which enables Ser51 to engage the phosphoacceptor binding site of the kinase. Finally, we propose that the protected state of Ser51 in free eIF2alpha prevents promiscuous phosphorylation and the attendant translational regulation by heterologous kinases. In order to subvert the anti-viral defense mediated by PKR viruses produce inhibitors of the kinase. Several members of the poxvirus family express two different types of PKR inhibitors: a pseudosubstrate inhibitor (such as the vaccinia virus K3L protein that resembles the N-terminal third of eIF2alpha) and a double-stranded RNA binding protein called E3L. High-level expression of human PKR inhibited the growth of yeast, and co-expression of the vaccinia virus K3L or E3L protein or the related variola (smallpox) virus C3L or E3L protein, respectively restored yeast cell growth. We previously identified PKR mutations that confer resistance to K3L inhibition but do not affect eIF2alpha phosphorylation. We proposed that these paradoxical effects on pseudosubstrate versus substrate interactions reflect differences between the rigid K3L protein and the plastic nature of eIF2alpha around the Ser51 phosphorylation site. We are currently characterizing PKR mutations that confer resistance to E3L inhibition. We have also characterized an eIF2alpha ortholog, termed vIF2alpha, found in ranaviruses that infect lower vertebrates including fish, frogs and salamanders. Our studies revealed that vIF2alpha does not functionally substitute for eIF2alpha in yeast. However, expression of vIF2alpha suppresses the toxic effects of PKR in yeast. We conclude that vIF2alpha functions as an inhibitor of PKR probably as a pseudosubstrate like vaccinia virus K3L. The gamma subunit of eIF2 is a GTPase that, based on sequence and the structure of the archaeal homolog aIF2gamma, resembles elongation factor EF-Tu. However, in contrast to EF-Tu, which binds to the A-site of the 70S ribosome, eIF2 binds Met-tRNAi to the P-site of the 40S subunit. To gain insights into how eIF2 binds Met-tRNAi and then associates with the 40S ribosome, we used directed hydroxyl radical probing to identify eIF2 contacts within the 40SeIF1eIF1AeIF2GTPMet-tRNAimRNA (48S) complex. Based on the structure of the EF-Tu ternary complex, we predicted that linkage of Fe(II)-BABE, a hydroxyl radical generator, to domain III of eIF2gamma would result in cleavage of Met-tRNAi in the T-stem. However, this instead resulted in cleavage of the D-stem of Met-tRNAi and of 18S rRNA at the top of helix h44, a prominent landmark on the intersubunit surface of the 40S subunit. Based on the results of these and other cleavage experiments, and the fact that Met-tRNAi is bound to the P-site of the 40S subunit, we generated a model of the 48S complex in which domain III of eIF2gamma binds near 18S rRNA helix h44 and eIF2gammaMet-tRNAi contacts are restricted to the acceptor stem of the tRNA. In this model of the eIF2 ternary complex, the Met-tRNAi is rotated nearly 180 degrees relative to the position of the tRNA in the EF-Tu ternary complex. Consistent with the alternate models of the eIF2 and EF-Tu ternary complexes, we found that a domain III mutation in EF-Tu severely impaired Phe-tRNA binding;whereas, the corresponding eIF2gamma mutation did not impair Met-tRNAi binding to eIF2. Thus, despite their structural similarity, eIF2 and EF-Tu bind tRNA in substantially different manners, and we propose that the tRNA-binding domain III of EF-Tu has acquired a new function in eIF2gamma to bind the ribosome. The GTP-binding protein eIF5B catalyzes ribosomal subunit joining in the final step of translation initiation. Our previous studies revealed that GTP hydrolysis by eIF5B activates a regulatory switch required for eIF5B release from the ribosome following subunit joining. The eIF5B resembles a chalice with the α-helix H12 forming the stem connecting the GTP-binding domain cup to the domain IV base. Helix H12 has been proposed to function as a rigid lever arm governing domain IV movements in response to nucleotide binding and as a molecular ruler fixing the distance between domain IV and the G domain of the factor. To investigate its function, we altered the length and rigidity of helix H12. Whereas helix H12 mutations had minimal impacts on GTP binding and on eIF5B ribosome binding and GTPase activities, shortening the helix impaired the rate of subunit joining in vitro and both shortening the helix and increasing its flexibility impaired the stability of Met-tRNA bound to the 80S product of subunit joining. These data support the notion that helix H12 functions as a ruler connecting the GTPase center of the ribosome to the P site where Met-tRNA is bound and that helix H12 rigidity is required to stabilize Met-tRNA binding. The translation factor eIF5A, the sole protein containing the unusual amino acid hypusine N-epsilon-(4-amino-2-hydroxybutyl)lysine, was originally identified based on its ability to stimulate the yield (endpoint) of methionyl-puromycin synthesis, a model assay for first peptide bond synthesis. However, the precise cellular role of eIF5A is unknown. Using molecular genetic and biochemical studies, we recently showed that eIF5A promotes translation elongation, and this activity is dependent on the hypusine modification. As eIF5A is a structural homolog of the bacterial protein EF-P, we propose that eIF5A/EF-P is a universally conserved translation elongation factor.

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Ivanov, Ivaylo P; Shin, Byung-Sik; Loughran, Gary et al. (2018) Polyamine Control of Translation Elongation Regulates Start Site Selection on Antizyme Inhibitor mRNA via Ribosome Queuing. Mol Cell 70:254-264.e6
Dever, Thomas E; Dinman, Jonathan D; Green, Rachel (2018) Translation Elongation and Recoding in Eukaryotes. Cold Spring Harb Perspect Biol 10:
Skopkova, Martina; Hennig, Friederike; Shin, Byung-Sik et al. (2017) EIF2S3 Mutations Associated with Severe X-Linked Intellectual Disability Syndrome MEHMO. Hum Mutat 38:409-425
Schuller, Anthony P; Wu, Colin Chih-Chien; Dever, Thomas E et al. (2017) eIF5A Functions Globally in Translation Elongation and Termination. Mol Cell 66:194-205.e5
Shin, Byung-Sik; Katoh, Takayuki; Gutierrez, Erik et al. (2017) Amino acid substrates impose polyamine, eIF5A, or hypusine requirement for peptide synthesis. Nucleic Acids Res :
Melnikov, Sergey; Mailliot, Justine; Rigger, Lukas et al. (2016) Molecular insights into protein synthesis with proline residues. EMBO Rep 17:1776-1784
Melnikov, Sergey; Mailliot, Justine; Shin, Byung-Sik et al. (2016) Crystal Structure of Hypusine-Containing Translation Factor eIF5A Bound to a Rotated Eukaryotic Ribosome. J Mol Biol :
Murray, Jason; Savva, Christos G; Shin, Byung-Sik et al. (2016) Structural characterization of ribosome recruitment and translocation by type IV IRES. Elife 5:
Dever, Thomas E; Kinzy, Terri Goss; Pavitt, Graham D (2016) Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae. Genetics 203:65-107
Rojas, Margarito; Vasconcelos, Gabriel; Dever, Thomas E (2015) An eIF2?-binding motif in protein phosphatase 1 subunit GADD34 and its viral orthologs is required to promote dephosphorylation of eIF2?. Proc Natl Acad Sci U S A 112:E3466-75

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