Summary Aminoacyl-tRNA synthetases (ARSs) establish the rules of the genetic code, whereby each amino acid is attached to a cognate tRNA. Errors in this process lead to mistranslation, which can be toxic to cells. Recent studies suggest that the selective forces exerted by cell-specific requirements and environmental conditions potentially shape quality control mechanisms. Approximately half of the ARSs possess a proofreading (or editing) function to hydrolyze mischarged aa-tRNAs and evidence that non-proteinaceous amino acids pose the greatest threat to fidelity is beginning to emerge. Early work in the Musier-Forsyth lab in the field of translational quality control focused on our discovery of Class II prolyl-tRNA synthetase (ProRS) editing. This led to a detailed mechanistic understanding of the novel bacterial ProRS posttransfer editing domain (INS) and the demonstration that the INS domain, when purified on its own outside the context of the ARS, was fully functional in tRNA deacylation. We subsequently discovered that single-domain INS homologs are widespread in Bacteria and in recent years, our focus in this area has turned almost entirely to understanding the function of these INS-like domains in tRNA editing. However, many open questions regarding the physiological function of these putative trans-editing proteins remain. The overarching goal of the research described in this MIRA application is to uncover the specific functions of a growing family of trans-editing proteins known as the INS superfamily. This diverse yet universally conserved family now has a solid and accumulating in vitro structure-function knowledge base, which strongly supports a role in maintaining translational fidelity. Our knowledge of the broader physiological roles of these proteins, especially in eukaryotes, is still in its infancy and is just beginning to reveal wider roles than previously anticipated. This major gap will be addressed in this work. While classical knock- down screens that only define essential versus non-essential genes do not immediately identify editing domains as essential, the strong conservation of these domains implies they play important, and in most cases still undiscovered, roles in cell survival and competitiveness. Proposed studies are designed to address some of the many open questions with regard to both physiological trans-editing functions and potential moonlighting functions of the INS superfamily. These domains are largely unexplored in eukaryotes, including a novel sub- family cluster that is encoded in many unicellular eukaryotic pathogens. The therapeutic potential of trans-editing domains has not been exploited and represents another major gap in the field that we hope to address by our planned studies. In the long term, combining drugs that target novel translational fidelity mechanisms along with known ribosome-targeting protein synthesis inhibitors such as aminoglycosides, may results in more effective therapeutic strat...