Genetic Approaches to Protein-Nucleic Acid Interactions

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DESCRIPTION (provided by applicant): This project is focused on how components of the translation apparatus--aminoacyl tRNA synthetases-are connected to biological pathways outside of translation and to disease. The synthetases catalyze aminoacylation of transfer RNAs and thereby establish the genetic code relationship between trinucleotide codons and amino acids. The enzymes appeared early in evolution and are essential to all life forms. Small errors in aminoacylation result in mistranslation, that is, the incorporation of the wrong amino acid into a growing polypeptide chain. These errors are normally corrected by editing activities of synthetases, whereby a mischarged amino acid is cleared from the tRNA to which it is attached. The editing sites in synthetases are highly conserved in evolution, and this conservation speaks to their being essential. Because of the build up over time of proteins containing errors, mutations that disrupt editing are toxic to bacteria. Mammalian cells were shown in the last grant period to be far more sensitive than bacteria to errors of aminoacylation. Cells harboring an editing defect are driven into an apoptosis-like response in a trans-dominant way. In addition, a mild mutation in the activity of a specific tRNA synthetase was shown to cause neurodegeneration in mouse that, among other phenotypes, resulted in ataxia. This mild editing defect generated strong upregulation of the unfolded protein response and caused degeneration of Purkinje cells of the cerebellum. Further work showed that editing defects are mutagenic in aging bacteria, because of mistranslation of proteins in the DNA replication apparatus, including the DNA repair systems. Moving forward on the foundation of these recent results, future experiments are designed to investigate how editing defects can lead to the development of cancer (oncogenic transformation) in aging cells. Other work is aimed at understanding a key mechanism by which mammalian cells overcome their struggle to avoid confusing serine and glycine for alanine. This confusion is dealt with, in part, by an editing-proficient genome-encoded fragment that is homologous to the editing domain of a specific synthetase, and that can act in trans to clear a tRNA that is mischarged with serine or glycine instead of with alanine. Although the activity of the editing-proficient genome-encoded fragment has been demonstrated in vitro, the significance of the activity in vivo remains to be established and is a goal of the next grant period. In addition, this fragment is fused to a molecular co-chaperone. How the co-chaperone moiety of the fusion regulates the editing activity, and how the fusion connects the editing activity to other biological systems, is of great interest in the future program. PUBLIC HEALTH RELEVANCE: This project is focused on how errors in protein synthesis lead to cell death and disease. The emphasis is on understanding a key mechanism for error-avoidance and the kinds of diseases that can result when this mechanism breaks down.