Roles of Human elF1 in Translation Preinitiation Complex Assembly

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DESCRIPTION (provided by applicant): Based on studies in yeast Saccharomyces cerevisiae, the eukaryotic translation initiation factor (elF) 5 has been shown to serve a critical role in the assembly of an important intermediate in the 43S pre-initiation complex via the formation of multiple binding interfaces mediated by its carboxyl terminal domain (elF5-CTD). This grant proposes investigations to determine the structural mechanisms involved in the formation of the human elF1/elF5-CTD complex, which is an important step in the assembly of the translation pre-initiation complex. Objective/Hypothesis: Preliminary studies in our laboratory indicate that elF1 and elF5-CTD interact and form a complex. Utilizing NMR spectroscopy, we are currently determining the mutual binding interface of the elF1/elF5-CTD complex. This grant proposes investigations to determine the multiple binding-interfaces of the elF1/elF5-CTD complex with components of the human pre-initiation complex. This novel complex mediates an important step in the eukaryotic translation initiation process. In order to investigate the structural role of the elF1/elF5-CTD sub-complex within the pre-initiation complex, we will address the following specific aims: 1.) Map the mutual binding interface of the human elF1/elF5 complex and determine its structure. 2.) Characterize the interaction of human elF5-CTD and elF1/elF5- CTD complex with elF2-beta and elF4G. Study Design: In order to tackle the multiple binding-interface of the elF1/elF5-CTD complex, the scope of this grant will encompass the utilization of nuclear magnetic resonance (NMR) spectroscopy. We will optimize conditions for NMR chemical shift mapping of the multiple binding-interface of the elF1/elF5-CTD complex. An in vitro reconstitution system involving purified proteins in which one component is 15N-labeled (e.g. elF5-CTD) while in the presence of its unlabeled binding partners (e.g. elF1 and elF2-beta). The mapping of the multiple faces of the human elF1/elF5 complex will give an invaluable insight into the assembly the human translation pre-initiation complex. Insights into the structural interactions of the elF1/elF5-CTD sub-complex could lead to the future development of small molecule inhibitors to modulate human translation initiation leading to novel, robust pharmaceutical drugs to treat cancer.