Dynamic mechanisms of human mitochondrial translation - PROJECT SUMMARY Human mitochondria contain a dedicated translation system that produces 13 essential proteins required for oxidative phosphorylation and ATP generation. Disruption of this process contributes to a wide range of human diseases, including cancer, metabolic syndrome, cardiovascular disorders, and neurodegenerative conditions. Because of its bacterial origin, mitochondrial translation is also inadvertently targeted by commonly used antibiotics, leading to mitochondrial toxicity. Genetic, biochemical, and structural studies have identified the key components involved in mitochondrial translation and provided structural snapshots of mitoribosomal complexes, underscoring the importance of dynamic ligand–ribosome interactions. Together with ribosome profiling and functional cellular studies, these efforts have revealed numerous unique features of mammalian mitochondrial translation. Despite this progress, the dynamic mechanisms that govern mitochondrial protein synthesis remain poorly understood, hindering interpretation of pathogenic mutations and the design of safer antibiotics. Key challenges include the difficulty of manipulating the mitochondrial genome, limited access to homogeneous mitochondrial material, and the inherent complexity of translation, which involves transient intermediates and likely parallel pathways. In my postdoctoral research, I pioneered the use of in vitro reconstitution and single-molecule spectroscopy approaches to investigate the dynamic mechanisms of yeast cytosolic translation. These approaches enable direct, real-time observation of translation by tracking individual dye-labeled ribosomal subunits, mRNAs, tRNAs, and protein factors. Building on this expertise, my independent research group has developed a unique purified human mitochondrial translation system suited for single-molecule analysis. During the ESI MIRA phase, we will integrate single-molecule spectroscopy with complementary biochemical, structural, and cellular approaches to define the dynamic mechanisms of human mitochondrial translation. In Area 1, we will determine how mitoribosomes initiate translation on leaderless mRNAs and how initiation is regulated by mRNA structures and the LRPPRC–SLIRP complex. In Area 2, we will explore how structurally atypical mitochondrial tRNAs decode the mRNA codons and how polyproline- induced ribosome stalling is rescued by the mitochondrial-specific factor TACO1. In Area 3, we will define the dynamic processes that ensure accurate and efficient translation termination and ribosome recycling at canonical and non-canonical stop codons. By providing quantitative, high-resolution models of these essential steps, our work will establish a comprehensive framework for understanding human mitochondrial translation and its regulation in health and disease, informing the development of targeted therapies and safer antibiotics.
