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 the structure-function properties of these factors, and to define their interactions with the ribosome. We are also characterizing mutations in eIF2γ associated with a human X-linked intellectual disability called MEHMO syndrome. The GTPase eEF2 functions in the elongation phase of protein synthesis to promote translocation of the tRNAs and mRNA on the ribosome following each round of peptide bond formation. We are using biochemical and molecular approaches to investigate the role of the novel diphthamide modification on eEF2.
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 and other eIF2α kinases 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 eIF2α kinase 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. We are also studying the mechanisms by which phosphorylation of eIF2α enhances translation of mRNAs encoding key stress responsive factors.
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]. Using molecular-genetic and biochemical approaches, we showed that eIF5A promotes translation elongation and that this activity is dependent on the hypusine modification. We also showed that eIF5A, like its bacterial ortholog EF-P, stimulates the peptidyl-transferase activity of the ribosome and facilitates the reactivity of poor substrates such as proline. Accordingly, eIF5A and its hypusine modification are required both in vivo and in vitro for the synthesis of proteins containing polyproline sequences. Together with Marat Yusupov we obtained the structure of eIF5A bound to the yeast 80S ribosome and we are characterizing translational control mechanisms that exploit the eIF5A-dependency for polyproline synthesis. Studies of antizyme inhibitor (AZIN1) translational control revealed a novel mechanism of translation start site selection involving ribosome queuing. Whereas most ribosomes bypass an upstream Conserved Coding region (uCC) in the AZIN1 mRNA due to the lack of an AUG start codon, an occassional ribosome translates the uCC by initiating at a near cognate start codon. Under conditions of high polyamines, which inhibit eIF5A function, ribosomes elongating on the uCC pause at a proline motif. Subsequent scanning ribosomes form a queue behind the paused elongating ribosome. This queue positions a scanning ribosome over the uCC start site and enhances initiation on the uCC, which leads to repression of AZIN1 synthesis. Ongoing studies are identifying and characterizing additional mRNAs whose translation is regulated by a uCC-type mechanism.