Ribosomes are large ribonucleoprotein complexes, which interact with a number of protein factors and RNA molecules to carry out the essential process of protein biosynthesis, or translation, in all cells, and also within organelles, such as mitochondria, in eukaryotic cells. An understanding of the structural and functional differences between bacterial and mitochondrial ribosomes (mitoribosomes) must underpin the treatment of bacterial infectious diseases. While bacterial ribosomes are the targets of several antibiotics, it is essential that the mitoribosomes of the host cell not be susceptible to the effects of the antibiotic (toxicity), if the synthesis of polypeptides required for the production of most of the cell's energy is to be safeguarded. Furthermore, defects in mitochondrial translation are associated with several human mitochondrial diseases. The long-term goal of our study is to understand the mechanism of translation in mammalian mitochondria, by determining structures of various functional complexes of the mitoribosome. This knowledge will facilitate the identification of drug targets specific to the bacterial system. Also, an improved understanding of the functioning of the mammalian mitoribosome will let us begin to understand the dysfunctions related to mitochondrial translation. Structures for specific functional complexes of mammalian mitoribosomes will be determined by 3D cryo- electron microscopy (cryo-EM). Mammalian mitoribosomes are inherently poor candidates for crystallographic analysis due to their compositional heterogeneity, and low abundance in the cell, making cryo-EM one of the few tools available to obtain structural information on these complex particles. Four functional aspects of the mitoribosome will be studied. (1) To study initiation, the structures of mitochondrial translation initiation complexes involving initiation factors IF2mt and IF3mt and the small 28S mitoribosomal subunit will be determined. (2) To study elongation, ongoing studies of elongation complexes involving elongation factor, EF- G1mt will be continued. (3) To study recycling, structures for mitoribosome complexes with the ribosome recycling factor, RRFmt, in the presence and absence of elongation factor, EF-G2mt, will be determined. (4) To examine the interaction of the mitoribosome with Oxa1L, a protein known to interact with nascent polypeptide chains and to aid in their co-translational insertion into the mitochondrial inner membrane, structures of complexes between the mitoribosome and the Oxa1L protein will be determined. The cryo-EM maps from each of these four areas will be analyzed with docking methods, utilizing atomic structures and homology models of the mitoribosome and the respective protein factors. These studies will allow a direct comparison of specific steps of translation between the bacterial system, for which structures of homologous complexes are readily available, and the mammalian mitochondrial system, and will lead to the identification of potential drug targets.
The mammalian mitochondrial ribosome is responsible for synthesizing polypeptide chains that are involved in the generation of more than 90% of the energy required by the cell. The proposed study will help develop an understanding the structure and function of the mammalian mitochondrial ribosome which synthesizes these proteins. This information will help (i) increase our understanding the human genetic diseases that are caused by defects in mitochondrial protein synthesis, and (ii) facilitate the design of more specific drugs to target bacterial protein synthesis.
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