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.
Changes in the activity or level of the eukaryotic Translation Elongation Factor 1 (eEF1A) protein or drugs that target its activity can affect not only how proteins are made in the cell but also the ability of a virus to replicate and cellular organizatio. Studies have shown that the activity of this protein is altered in some cancers and during aging but it is not clear which of its many functions play a role in these processes. How these diverse functions of eEF1A are connected and regulated, and how cellular organization effects protein synthesis, are poorly understood.
|Dever, Thomas E; Kinzy, Terri Goss; Pavitt, Graham D (2016) Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae. Genetics 203:65-107|
|Sasikumar, Arjun N; Kinzy, Terri Goss (2014) Mutations in the chromodomain-like insertion of translation elongation factor 3 compromise protein synthesis through reduced ATPase activity. J Biol Chem 289:4853-60|
|Perez, Winder B; Kinzy, Terri Goss (2014) Translation elongation factor 1A mutants with altered actin bundling activity show reduced aminoacyl-tRNA binding and alter initiation via eIF2Î± phosphorylation. J Biol Chem 289:20928-38|
|Sasikumar, Arjun N; Perez, Winder B; Kinzy, Terri Goss (2012) The many roles of the eukaryotic elongation factor 1 complex. Wiley Interdiscip Rev RNA 3:543-55|
|Visweswaraiah, Jyothsna; Lageix, Sebastien; Castilho, Beatriz A et al. (2011) Evidence that eukaryotic translation elongation factor 1A (eEF1A) binds the Gcn2 protein C terminus and inhibits Gcn2 activity. J Biol Chem 286:36568-79|
|Sasvari, Zsuzsanna; Izotova, Lara; Kinzy, Terri Goss et al. (2011) Synergistic roles of eukaryotic translation elongation factors 1BÃ½Ã½ and 1A in stimulation of tombusvirus minus-strand synthesis. PLoS Pathog 7:e1002438|
|Mateyak, Maria K; Kinzy, Terri Goss (2010) eEF1A: thinking outside the ribosome. J Biol Chem 285:21209-13|
|Li, Zhenghe; Pogany, Judit; Tupman, Steven et al. (2010) Translation elongation factor 1A facilitates the assembly of the tombusvirus replicase and stimulates minus-strand synthesis. PLoS Pathog 6:e1001175|
|Esposito, Anthony M; Mateyak, Maria; He, Dongming et al. (2010) Eukaryotic polyribosome profile analysis. J Vis Exp :|
|Van Goietsenoven, Gwendoline; Hutton, Jenna; Becker, Jean-Paul et al. (2010) Targeting of eEF1A with Amaryllidaceae isocarbostyrils as a strategy to combat melanomas. FASEB J 24:4575-84|
Showing the most recent 10 out of 34 publications