We study the mechanism and regulation of protein synthesis in eukaryotic cells. Of special interest are the regulation of protein synthesis by GTP-binding (G) proteins and protein phosphorylation. In addition, we are studying unusual post-translational modifications of the factors that assist the ribosome in synthesizing proteins as well as the mRNA features and translational components that mediate the fidelity of translation start site selection. The first step of protein synthesis is binding the initiator Met-tRNA to the small ribosomal subunit by the factor eIF2. The eIF2 is composed of three subunits including the G protein eIF2gamma. During translation initiation, the GTP bound to eIF2gamma is hydrolyzed to GDP, and the factor eIF2B recycles eIF2-GDP to eIF2-GTP. Phosphorylation of eIF2alpha on serine 51, by a family of stress-responsive protein kinases, coverts eIF2 into an inhibitor of eIF2B and triggers the integrated stress response (ISR). Our recent studies on eIF2 have provided insights into the human disease MEHMO syndrome. Protein synthesis plays a critical role in learning and memory in model systems, and our studies have linked a human X-linked intellectual disability (XLID) syndrome to altered function of eIF2. In previous studies, with collaborators from multiple countries, we showed that MEHMO syndrome, a human XLID syndrome with additional symptoms including epilepsy, hypogonadism and hypogenitalism, microcephaly, and obesity as well as hypopituitarism and hypoglycemia is caused by mutations in the EIF2S3 gene encoding the gamma subunit of eIF2. Over the past year we have been characterizing additional novel EIF2S3 mutations identified in patients with MEHMO syndrome. This work complements our previous studies characterizing other EIF2S3 mutations linked to MEHMO syndrome and will further our understanding of the molecular defects in eIF2 that cause the varied symptoms of the disease. In previous studies we used induced pluripotent stem (iPS) cells derived from a patient with MEHMO syndrome. Our studies revealed defects in general protein synthesis, constitutive induction of the ISR, and hyper-induction of the ISR under stress conditions The EIF2S3 mutation also impaired neuronal differentiation by the iPS cells. Interestingly, the drug ISRIB, an activator of the eIF2 guanine nucleotide exchange factor, rescued the cell growth, translation, and neuronal differentiation defects associated with the EIF2S3 mutation, offering the possibility of therapeutic intervention for MEHMO syndrome. Our current efforts are aimed at generating a mouse model of MEHMO syndrome. A second major focus is the translation factors eIF5B, a GTPase required for the last step of translation initiation joining of the large ribosomal subunit to small subunit poised on the start codon of an mRNA and eEF2, a GTPase that promotes translation elongation. In collaboration with researchers at Stanford, we helped show that eIF5B reorients the initiator Met-tRNAi to enable joining of the large ribosomal subunit. Our studies on the diphthamide modification of eEF2 revealed a critical role in reading frame maintenance including on the programmed frameshift site in SARS-CoV-2. Selection of the translation start site (typically an AUG codon) in eukaryotes is influenced by context nucleotides flanking the AUG codon and by levels of the factors eIF1 and eIF5. In our third major focus, we conducted a search of mammalian genes and identified five homeobox (Hox) gene paralogs initiated by AUG codons in conserved suboptimal context as well as 13 Hox genes that contain evolutionarily conserved upstream open reading frames (uORFs) that initiate at AUG codons in poor sequence context. Our collaborators at the Johns Hopkins University mapped the 5 end of the Hox mRNAs, revealing that the mRNAs are much shorter than previously reported and lack proposed alternative translation elements. We found that the conserved uORFs inhibit Hox reporter expression and that altering the stringency of start codon selection by overexpressing eIF1 or eIF5 modulates the expression of Hox reporters. We also show that modifying ribosome homeostasis by depleting a large ribosomal subunit protein or treating cells with sublethal concentrations of puromycin lowers the fidelity of start codon selection. As the Hox genes encode developmental regulators of animal body plans, our findings reveal that alteration in start codon selection stringency has the potential to regulate global gene expression programs, including Hox gene-directed body plan formation in animals. A fourth major focus is the translation factor eIF5A and polyamines. The eIF5A is the sole cellular protein containing the unusual amino acid hypusine. Using molecular genetic and biochemical studies, we previously showed that eIF5A promotes translation elongation, and that this activity is dependent on the hypusine modification. Moreover, certain amino acid motifs like runs of consecutive proline residues showed a heightened dependency on eIF5A for their translation both in cells and in vitro. Based, in part, on our studies with x-ray crystallographers in France, we propose eIF5A and its hypusine residue function to reposition the acceptor arm of the P site tRNA on the ribosome to enhance reactivity towards either an aminoacyl-tRNA, for peptide bond formation, or a release factor, for translation termination. Interestingly, the hypusine modification on eIF5A is derived the polyamine spermidine, and we have discovered several connections between eIF5A and polyamines. We continue to study the link between eIF5A and the regulation of polyamine metabolism in mammalian cells. The enzyme ornithine decarboxylase (ODC) catalyzes the first step in polyamine synthesis. ODC is regulated by a protein called antizyme (OAZ), which, in turn, is regulated by another protein called antizyme inhibitor (AZIN). The synthesis of OAZ is stimulated by polyamines while AZIN synthesis is inhibited. The regulation of AZIN synthesis is dependent on a conserved uORF in its mRNA. Our data indicate that polyamines interfere with eIF5A binding on the ribosome, triggering a ribosome pause that governs translation of the inhibitory uORF on the AZIN mRNA and thereby represses AZIN synthesis. We have also linked polyamine inhibition of eIF5A to stimulation of ribosomal frameshifting on the OAZ mRNA and to uORF-mediated inhibition of S-adenosylmethionine decarboxylase (AMD1) synthesis. Our studies thus identify eIF5A as a general sensor and effector for autoregulation of polyamine biosynthesis. Finally, we previously showed that Hol1 is the yeast high-affinity polyamine transporter. Together with Anirban Banerjees lab in the NICHD, we showed that purified Hol1 transports polyamines, and we found that polyamine inhibition of the translation factor eIF5A controls translation of the HOL1 mRNA. Thus, polyamine transport, like polyamine biosynthesis, is under translational autoregulation by polyamines in yeast, highlighting the extensive control cells impose on polyamine levels. In ongoing studies, we are characterizing the HOL1 orthologs in the pathogenic yeast Candida albicans, and we are performing mutagenic structure-function studies on HOL1 to understand how the protein specifically recognizes and transports polyamines.