The broad objective of this work is to contribute to our fundamental understanding of how high fidelity in protein synthesis is achieved during translation of the genetic code. Transfer RNA recognition by class II aminoacyl-tRNA synthetases (aaRS) is a key feature of this process and is one focus of this work. There is considerable variation in the mode of tRNA recognition and discrimination even within the same subclass of synthetases. Thus, it is important to study in detail each of the twenty systems, as each will provide new insights into molecular interactions responsible for the accurate translation of the genetic code. The tRNA synthetases represent a classic problem in molecular recognition and discrimination that is a paradigm for all types of similar recognition events. Therefore, the principles gained from these studies may be applied to a wide variety of RNA-protein interactions that play crucial roles in gene expression and regulation. The mechanism by which aaRSs discriminate among structurally related amino acids is another major focus of this work. In particular, the amino acid editing function of prolyl-tRNA synthetases (ProRS) is being investigated. We have shown that the YbaK protein, a paralog of the ProRS editing domain, which is present in all three kingdoms of life, can also carry out editing of misacylated tRNApro species in trans. This novel mechanism of editing may be exploited for new antibiotic development, however, additional studies are needed to explore the mechanism of editing as well as species-specific differences in this activity. Therefore, the specific aims of this work are: (1) To explore species-specific differences in tRNA recognition by class II ProRS and histidyl-tRNA synthetase (HisRS) and to understand how these protein-RNA interactions have co-adapted to accommodate evolutionary changes, (2) to explore the mechanism of amino acid editing by ProRS, and (3) to explore the hydrolysis activity and substrate specificity of the YbaK/ProX family of proteins.
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