Recently, the structural basis of protein synthesis in prokaryotes has been illuminated by a series of structures of the soluble translation factors and the ribosome itself. A similar understanding of the more complicated and highly regulated steps of eukaryotic protein synthesis is just beginning. The goal of this project is to understand the structural basis for the function and regulation of the eukaryotic translation Elongation Factor 1A (eEF1A). eEF1A is a prototypic G-protein that performs functions not only in translation but also tRNA export, viral replication and cytoskeletal organization. Thus, it is clear that the cell needs to modulate the activity or levels of eEF1A for normal cellular growth. The regeneration of active eEF1A by the guanine nucleotide exchange factor eEF1B is illuminated by our structure of Saccharomyces cerevisiae eEF1A complexed with the catalytic fragment of the eEF1Balpha subunit. We propose to produce structures of eEF1A in complex with 1) GDP or the GTP analogue GDPNP 2) aminoacyl-tRNA (aa-tRNA) and GDPNP, 3) the complete guanine nucleotide exchange factor eEF1B and 4) actin. As a G-protein, eEF1A switches between active and inactive forms The based on whether GDP or GTP is bound. Hence, understand the structural switch between the forms will help elucidate the regulation of binding to the ribosome and aa-tRNA. The GTP form binds aa-tRNA, and the structure of this complex will illuminate the overall tertiary structure of the complex that binds the ribosomal A-site and senses a proper codon-anticodon interaction. The structure of the entire eEF1AB complex will help determine the function of the eEF1Bgamma subunit, a protein highly conserved in all eucaryotes but only recently implicated in perhaps modulating translational accuracy and the stress response. Lastly, the growing evidence of alternative functions of the translational apparatus, and in particular the ability of eEF1A to bind and bundle actin, leads us to expand out analysis to understand the structural basis of this association. With S. cerevisiae, the structural information obtained on eEF1A can be utilized for molecular genetic analysis of the critical residues for function and regulation.
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