We propose investigating basic principles of eukaryotic translation initiation using molecular biology and nuclear magnetic resonance techniques. Regulation of translation is a crucial aspect of benign and malignant cell growth. In this proposal we plan to study proteins that are key factors of translation initiation, their interaction with mRNA and principles of the regulation of translation initiation as mediated by protein phosphorylation. The research will be focused on the eukaryotic translation initiation factor eIF4E, its interaction with the mRNA m7GpppX cap structure and with other proteins. The protein eIF-4E is a central factor of translation initiation; its importance for translation is comparable to that of the TATA binding protein (TBP) for transcription. It shows no homology to any protein with known 3D structure. Phosphorylation of initiation factors plays an important role in the regulation of translation initiation. For example, insulin-stimulated cell growth is related to phosphorylation (apparently via the MAP kinase pathway) of an elF-4E binding protein that will be studied in this project as well (4E- BP2). In the first phase of the proposed research the solution structure of elF4E will be solved, complexed with the minimum-size cap structures, m7GpppG and m7Gpp. The effect of phosphorylating Ser-53 on the structure and function of eIF-4E will also be studied. A family of recently identified eIF-4E binding proteins (4E-BP1, 4E-BP2 and PHAS-I) has been shown to inhibit translation initiation by binding to elF4E. Expression systems for these proteins of ca. 118 to 120 residues are available to us. The solution structures of one or several of these proteins will be pursued. Subsequently, we will study the binding of these proteins to eIF- 4E to investigate the mode of translation initiation. Phosphorylation of 4E-BP on Ser63 disables 4E-BP to bind elF-4E. Thus, the effect of phosphorylation on the conformation of 4E-BP will be investigated. On a broader scale, this research is aimed at elucidating the role of elF-4E for cell growth and cell transformation, and at understanding the mechanism of translation initiation in general. We expect that this research will be relevant to the understanding of tumorigenic uncontrolled cell growth.
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