Aminoacyl-tRNA synthetases (ARSs) establish the rules of the genetic code, whereby each amino acid (aa) 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 function to hydrolyze mischarged aa-tRNAs and evidence that non-protein aa metabolites pose the greatest threat to fidelity is beginning to emerge. Interestingly, single-domain proteins homologous to ARS editing domains are encoded in many genomes but many open questions regarding the physiological function of these putative trans-editing proteins remain. While proofreading of genetically-encoded aa's by ARSs is well documented, much less is known about how the translation quality control machinery prevents misincorporation of non- protein aa's. We hypothesize that tRNA-specific, as well as semi-promiscuous trans-editing proteins, which are capable of acting on a variety of tRNA substrates and whose expression can be regulated independent of the ARSs, have evolved to prevent both standard and non-protein aa mistranslation errors. We have identified a family of trans-editing factors collectively known as the INS superfamily, which ensures accurate and efficient translation of Pro and other codons. This family includes the cis-editing domain (INS) of bacterial prolyl-tRNA synthetase (ProRS), YbaK, and 4 ProXp's. While a subset of these proteins corrects ProRS-dependent errors, exciting preliminary studies have revealed distinct substrate specificities that extend beyond aa-tRNAPro and include non-protein aa's.
The specific aims of this work are: 1) To determine the mechanism of substrate recognition by bacterial ProXp-ala; 2) To reveal the trans-editing function of bacterial ProXp-abu in vitro and in vivo; and 3) To probe the physiological roles of Escherichia coli INS superfamily members.
The overarching objective of this work is to contribute to our fundamental understanding of how fidelity in protein synthesis is achieved through accurate substrate recognition by aminoacyl-tRNA synthetases (ARSs). Many ARSs and single domain ARS-like proteins use editing mechanisms to correct errors that, if left uncorrected, would lead to errors in protein synthesis. The widespread distribution of these editing domains in bacteria and the absence of most of these domains in eukaryotic cells suggest an avenue for the development of new antibacterial agents.
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