Control of gene expression at the level of translation is essential for cellular development, differentiation, growth and death. During translation, the rate-limiting step is the initiation phase where mRNA is unfolded and recruited to the 40s ribosomal subunit prior to scanning and elongation. In eukaryotes, the process is facilitated by the initiation factor complex eIF4F. This complex consists of eIF4E, the 25 kDa protein that binds mRNA at the 5' cap; eIF4A, a 45 kDa ATP dependent RNA helicase; and eIF4G a larger protein that recruits the entire complex, through interactions with eIF4E and eIF4A, to the 40s ribosomal subunit. Of the translation initiation factors, eIF4E is present in limiting amounts. Furthermore, the activity of eIF4E is modulated by the 4E-binding proteins (4E-BPs), which inhibit the assembly of eIF4F by preventing the binding of eIF4E with eIF4G, making this interaction a critical point of translational regulation. This idea is born out by the fact that overexpression of eIF4E causes transformation of cells in culture. Although eIF4E is not proven to be a primary oncogene in vivo, elevated levels of this protein are detected in breast cancers and tumor cells. This suggests that drugs designed to reduce the activity of eIF4E would be effective in controlling tumor growth. One strategy would be to design drugs that are capable of competing with eIF4G for the corresponding binding surface of eIF4E. To this end, we have initiated structural studies of yeast eIIF4E in complex with the corresponding binding domain of yeast eIF4G (residues 391-488 of eIF4GI-refeffed to as eIF4G-4EBD) using NMR spectroscopy. We have shown that e]IF4G-4EBD is unstructured alone and that an ordered conformation results upon binding to eIF4E. State-of-the art heteronuclear triple resonance experiments, optimized for larger proteins through the use of transverse-relaxation optimized spectroscopy (TROSY), have been performed on 90% 'H, U""""""""C, """"""""N labeled eIIF4G-4EBD in complex with unlabeled eIF4E. Backbone resonance assignments are currently underway.
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