The eukaryotic translation Elongation Factors (eEFs) are determinants of the accuracy and efficiency of protein synthesis. A growing body of evidence also supports the importance of translation elongation in the regulation of gene expression. Of the three eEFs in fungi, eEF1A is the GTP binding protein that delivers aminoacyl-tRNA (aa-tRNA) to the ribosomal A-site. It has become clear that eEF1A physically and functionally interacts with other cofactors such as a guanine nucleotide exchange factor, as well as proteins and processes outside translation elongation. Altered activity or levels of eEF1A are linked to important cellular phenotypes, as the protein is overexpressed in transformed cell lines and some cancers and increased expression results in susceptibility to transformation. Furthermore, two compounds have been recently identified that target the actin bundling activity of eEF1A, affecting the male reproduction system and inhibiting the growth of melanoma cells. The unifying theme of this proposal is that regulation of eEF1A affects the efficiency, accuracy and control of translation elongation. This regulation may occur through interactions with cofactors such as aa-tRNA and actin or modification by kinases. The three aims proposed break down the mechanistic analysis of eEF1A around classes of mutants that interrogate these key interactions and functions. Aim 1 utilizes eEF1A mutants that affect the multiple activities of eEF1A to address the mechanism by which altered eEF1A activity affects the initiation step of protein synthesis as evidenced by phosphorylation of a key regulatory initiation factor, eIF2. Linked with analysis of mutations that affect translational fidelity and nucleotide binding, these studies will provide new insights into how multiple aspects of translation may be linked, the mechanism and signal between activities of eEF1A and other cellular process, and how eEF1A alterations affect elongation. Aim 2 provides an analysis of the regulation of translation elongation and eEF1A activity via sites of phosphorylation. Recent high throughput MS data has provided a rich resource to address this question, and our preliminary results show the key role of these sites in vivo. Aim 3 utilizes state of the art technology to look at the status of all ribosome associated mRNAs with deep sequencing methods to understand the overlapping effects of alterations in the key activities of eEF1A on global gene expression. This proposal reflects the need to think between cellular processes, and will coordinate directed with global approaches to determine the breadth and specificity of the effects of altered activities of eEF1A from the perspective of translation.