The development of novel and effective treatments for viral infections requires a fundamental understanding of how viruses recruit the host cell translational machinery for viral protein synthesis during the early stages of infection. Understanding how large multi-subunit translation initiation factors and viral RNAs control the binding and activity of ribosomes poses a formidable challenge that forms the basis of this grant proposal. To tackle the difficult mechanistic and structural problems inherent in studying translation, we propose to launch a coordinated and interdisciplinary effort to determine the compositions, intermolecular interactions and structures of key initiation factor complexes and unravel the mechanisms that regulate protein synthesis in human cells and viruses. A key aspect of this P01 Program Project Grant will be to establish highly interactive collaborations across disciplines and research institutions to study the chemical, structural and mechanistic properties of key complexes controlling translation initiation. By combining the expertise of several investigators we propose to implement a battery of complementary biophysical and biochemical approaches including: Projects by Doudna and Cate, cryo-electron microscopy and X-ray crystallography of translation initiation factors and complexes; Project by Hershey, in vitro and in vivo translation assays with viral and cellular translation components; and Project by Sarnow, purification and analysis of viral translation complexes. A major component of this proposal will be the establishment of a core laboratory for mass spectrometry of large multi-subunit complexes that will serve as the nerve center and clearinghouse for analyzing the many protein and protein-RNA complexes involved in translation initiation. This core laboratory will be used extensively by all five Research Projects to identify the macromolecules and post-translational modifications that, respectively, comprise and control translation activity in human cells and viruses. Thus, our goal is to develop a highly synergistic and concerted effort to carry out a pioneering set of interdependent experiments by using overlapping molecular reagents but applying distinct and complementary research strategies to dissect the core machinery responsible for protein synthesis in normal and virally-infected mammalian cells. ? ?
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