Project Summary Transfer RNA (tRNA) is best known to function in ribosome-mediated protein synthesis. However, in a less known role, arginyl-tRNA is essential for catalyzing a unique and poorly understood protein post- translational modification, namely arginylation, that regulates protein turnover. In this arginylation reaction, ATE1 (Arginyltransferase 1) facilitates arginine transfer to protein targets using a mechanism that depends on, and is selective for, arginyl-tRNA(Arg) as the donor cofactor. ATE1-mediated protein arginylation was identified on hundreds of proteins and is recognized as a global regulator of eukaryotic cellular processes, including embryogenesis, stress responses, and aging. Deregulation of ATE1 is found in patients with Parkinson’s disease and metastatic prostate, liver, and skin cancers. Nonetheless, how ATE1 (and other aminoacyl-tRNA transferases) hijacks tRNA from the highly efficient ribosomal protein synthesis pathways and catalyzes the arginylation reaction remains a mystery. This proposal aims to elucidate the catalytic mechanism and regulation of ATE1-mediated protein arginylation in vitro and in cells. We will focus on interrogating the activity of ATE1 and the consequences of arginylation at three scales. Firstly, we will determine the molecular mechanisms ATE1 selects for arginyl-tRNA(Arg) and recognizes specific sites in protein targets through an integrative approach combining structural, biochemical, and biophysical methods. Once determined, this research will allow a better understanding of the growing classes of aminoacyl-tRNA transferases in general. Secondly, we will quantitatively determine the consequences of arginylation on target protein turnover in living cells. Protein degradation usually depends on poly-ubiquitination, a downstream or concurrent event following arginylation, and through either proteasomal or autophagy-lysosomal pathways. By examining specific model substrates for proteasome or autophagosome under normal or stressed conditions, we will determine the crosstalk between arginylation and ubiquitination; delineate the contribution of each degradation pathway; reveal the kinetics in cells. Lastly, we will investigate whether and how core components of the ribosomal translation machinery and nutrients affect protein arginylation. Mechanistically, these studies will expand our knowledge of the regulatory roles of amino acids and tRNAs, enrich our toolbox to study macromolecule regulation by tRNA-dependent aminoacylation, and reshape how we consider the functions of the charged tRNAs beyond protein synthesis. Together, this research provides fundamental knowledge about arginylation, lays the groundwork for discovering novel therapeutic strategies by modulating ATE1 activity and protein arginylation in Parkinson’s disease and metastatic cancers, and enables us to build integrative platforms for future research.