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Our studies of protein synthesis are focused on three main areas:
The GTPases eIF2 and eIF5B promote, respectively, the first and final steps in translation initiation. eIF2 forms a ternary complex with GTP and Met-tRNAiMet and binds Met-tRNAiMet to the small ribosomal subunit in the first step of translation initiation. After the small ribosomal subunit complex binds to an mRNA and via scanning selects the translation start site, eIF5B promotes joining of the large ribosomal subunit. Following GTP hydrolysis and release of eIF5B, the ribosome is poised to elongate and synthesize the protein encoded by the mRNA. Together with Stephen Burley we obtained the crystal structures of eIF5B and eIF2γ, the GTP-binding subunit in the eIF2 complex. We are using molecular genetic, biochemical and structural probing assays to further investigate the functions of eIF2 and eIF5B, to characterize their structure-function properties, and to define their interactions with the ribosome.
We are studying how the function of eukaryotic translation initiation factor 2 (eIF2) is regulated by phosphorylation, focusing on determinants of substrate recognition by the eIF2α protein kinases and the structure and regulation of these enzymes. eIF2 plays a pivotal role in the initiation of protein synthesis by delivering tRNAiMet to the ribosome, and also functions in recognition of the AUG start codon. Down-regulation of eIF2 by phosphorylation of its α-subunit is a highly conserved mechanism for inhibiting general protein synthesis under starvation and stress conditions in mammalian cells, and mediates gene-specific translational induction of GCN4 expression in yeast. The human eIF2α kinase PKR has growth suppressive properties and its induction is an important aspect of the interferon-mediated antiviral response. As a consequence, many viruses encode antagonists of PKR. We have reconstituted in yeast cells the antagonism of PKR by viral inhibitors and we are using yeast and mammalian cell experiments, as well as biochemical assays and evolution studies, to identify the molecular determinants of PKR regulation. In addition, having collaborated with Frank Sicheri at the Samuel Lunenfeld Research Institute in Toronto to obtain the crystal structure of PKR in complex with eIF2α, we are using molecular genetic and biochemical approaches to study the structure-function properties of PKR.
The factor eIF5A was originally identified by its ability to stimulate the yield (endpoint) of methionyl-puromycin synthesis, a model assay for first peptide bond synthesis. eIF5A is the only protein in eukaryotes and archaea containing the unusual amino acid hypusine [Ne-(4-amino-2-hydroxybutyl)lysine]. However, the precise cellular function of eIF5A and of hypusine is unknown, and eIF5A has also been linked to mRNA decay and to nucleocytoplasmic transport. Recently we showed using molecular genetic and biochemical studies that eIF5A promotes translation elongation, and current studies are aimed at further defining the role of eIF5A in protein synthesis.