PROJECT ABSTRACT Ribonucleic acids (RNAs) are amongst the most functionally versatile biomolecules found in nature, accomplishing diverse biological tasks including carrying genetic information (mRNA, viral genomes), catalyzing reactions (rRNA, RNase P, telomerase, group I/II introns), acting as molecular adaptors (tRNA), and regulating gene expression (riboswitches, lncRNA, miRNA). Alterations in the structure and dynamics of RNA molecules are linked to bacterial pathogenesis, cardiovascular conditions, neurological disorders, mitochondrial diseases, and cancers. Despite the biological importance of RNA, our understanding of fundamental aspects of RNA structure, function and biochemical properties lags far behind that of proteins. My research program studies how the structure and dynamics of mitochondrial transfer RNAs (mt-tRNAs) impact their maturation and function. Our work will fill long-standing knowledge gaps in RNA biology–including identifying molecular level consequences of disease causing mt-tRNA mutations (Aim 3), establishing the impact of discrete tRNA post-transcriptional modifications (Aims 1&2), and determining the high-resolution structures of a broad set of RNAs and the proteins involved in their maturation (Aims 1-3). Additionally, we will develop broadly applicable chemical tools for studying mitochondrial RNA biology (Aim 3). Our preliminary work suggests that mt-tRNAs have unique structural and biophysical properties, are often more dynamic and unstable prior to processing and modification, and, therefore, have unique maturation pathways. We posit that disease-causing mutations destabilize mt-tRNA structure and alter its ability to be recognized as a substrate by processing and modification enzymes, therefore affecting mt-tRNA biogenesis and therefore mitochondrial function. In this work we propose a combination of biochemistry (kinetic and binding assays), structural biology (Cryo-EM and X-ray crystallography), innovative bio-analytical characterization (IM-MS), biophysical tools (smFRET, NMR), chemical biology (high-throughput assays) and cellular biology to take on questions regarding how mt-RNA structure and mt-PRORP function contribute to mitochondrial health and pathology. These studies will reveal how tRNAs are recognized and processed by mt-PRORP and help rationalize how patient-derived mt-tRNA mutations alter mt-tRNA stability and processing, improving our understanding of fundamental aspects of mt-tRNA molecular biology and opening new avenues of exploration into the role of mitochondrial post-transcriptional RNA modification in mitochondrial diseases.