UC San Diego

Mitochondria are essential organelles within a eukaryotic cell, with their own genome (mtDNA) responsible for cellular energy production through oxidative phosphorylation. The mtDNA-encoded mitochondrial transfer RNAs (mt-tRNAs), despite comprising only a small fraction of the mitochondrial genome, account for a disproportionate number of disease-associated mutations. Many of these mutations disrupt mitochondrial gene expression and lead to severe mitochondrial disorders. However, the molecular mechanisms by which mt-tRNA sequence variation affect transcription and RNA maturation are insufficiently characterized. Understanding how these mutations alter the mt-tRNA life cycle is key to identify early points of dysfunction and for development of targeted therapeutic strategies. The goal of this dissertation is to define how mt-tRNA sequence variation influences transcriptional pausing on mt-tRNA encoding sequences and mt-tRNA processing, to establish mechanistic links between these processes and mitochondrial disease.Chapter 1, which serves as an introduction to the dissertation, provides an overview of mitochondrial transcription machinery and mt-tRNA processing, with the emphasis on the role of mt-tRNAs in coordinating RNA maturation. In Chapter 2, I investigate how disease-associated mutations within the acceptor stem of mt-tRNATyr impair 5ꞌ leader processing, disrupt interactions with the MRPP1/2 processing complex, and support a directional 5ꞌ-to-3ꞌ maturation pathway. This work further demonstrates that defects in upstream mt-tRNAs can propagate to downstream mt-tRNAs within clusters, revealing coordinated processing dependencies. Chapter 3 describes the development and optimization of the recombinant expression and purification strategies for key components of the human mitochondrial transcription machinery, including POLRMT, TFAM, and TFB2M. Through this work, I participated in establishing a robust biochemical platform for studying mitochondrial transcription in vitro, enabling mechanistic investigations of transcriptional regulation. In Chapter 4, I study transcriptional pausing by POLRMT and demonstrate that a disease-associated mutation (m.T616C) within mt-tRNAPhe abolishes a sequence-dependent pause. Through scaffold-based transcription assays, I show that this mutation disrupts transcriptional dynamics, linking mtDNA sequence variation to early defects in the mt-tRNA life cycle and suggesting a role for pausing in coordinating RNA folding and downstream processing. Overall, my work establishes a mechanistic framework connecting mt-tRNA sequence variation to defects in transcriptional pausing and coordinated mt-tRNA maturation. These findings provide new insight into how disease-associated mutations disrupt mitochondrial gene expression and highlight early stages of the mt-tRNA life cycle as critical points of vulnerability.