Generation of new mutants of eIF5A protein using the strategy of alanine scanning

Abstract

The translation initiation factor 5A (eIF5A) is a highly conserved protein in archae and eukaryotic cells and essential for cellular viability. This factor is the only cellular protein that undergoes an essential posttranslational modification dependent on the polyamine spermidine, called hypusination. Although this protein was originally identified as a translation initiation factor, subsequent studies did not support a role for eIF5A in general translation initiation. eIF5A has also been implicated in nuclear export of HIV-1 Rev and mRNA decay, but these findings are controversial in the literature and may reflect secondary effects of eIF-5A function. Recent evidence in favor of reconsidering the role of eIF5A as a translation factor and this data shown that eIF5A has a function more specifically in translation elongation. To improve understanding and description of the mechanism of eIF5A in translation, it is necessary to determine the connection between eIF5A and the ribosome and what kind of ribosomal complex eIF5A binds. Recent results from our laboratory have shown a direct binding of yeast eIF5A only in the large subunit of the ribosome, and the hypusine is required for this interaction. To characterize the interaction points between eIF5A and the translational machinery it is proposed the generation of mutants through the strategy of "charge cluster-to-alanine scanning". Later co-purification assays in vivo and in vitro to characterization of important residues for the interactions with the ribosome will be realized. The results of this project will contribute significantly to the characterization of the specific mechanism by which eIF5A affects protein synthesis. (AU)