The protein synthesis machinery, the ribosome, is not constructed of freely diffusing macromolecules; rather, its formation is an intricate and well-defined hierarchical process involving hundreds of proteins and a few RNA molecules working in cooperation and under tight regulation. In mitochondria, this fundamental process has an additional level of complexity, as it requires cooperative effort involving the precise regulation of two genomes. Although mitochondrial rRNAs are encoded in the mitochondrial genome, all 82 proteins of mitochondrial ribosome (mitoribosome) and numerous assembly factors are encoded in the nuclear genome and are therefore imported from cytosol. The high complexity of mitoribosomal assembly, and the unique association of a tRNA as a structural component of mitoribosome imply the involvement of as-yet-unknown mitochondria-specific auxiliary factors. Because many of the features of this system are unique to mitochondria, which have traditionally been difficult to investigate, little is known about the process of mitoribosome assembly.
I expected to reveal mechanistic insights into how mitoribosomal proteins are assembled in a cascade while nascent mitochondrial rRNA molecules are processed and folded. Since high resolution cryo-EM allows now a unique ability to investigate heterogeneous ribosomal populations and built de novo models, the proposed work will not only reveal maturation states, but also new factors in the mitoribosome assembly process.
Overall goal was to describe the full molecular sequence of the maturation steps along the ribosomal assembly pathway time line in human mitochondria and to put that sequence in the mirochondrial-cellular context. The objectives aimed to act on the fundamental question of the mitochondrial protein synthesis machinery assembly and uncover first insights into how unprecedented structural occurrences, unique to the ribosomes from human mitochondria, are formed.
During the period, in total of 6 assembly intermediate states from human mitochondria were identified by extensive 3D classifications of cryo-EM data and the model fitting is going on. In additon, two pre-initiation complexes of translation were also identified and published.
