Dynamic Behavior of Ribosome Ligands during Translation

Wadsworth Center, Menands NY

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Abstract

[unreadable] DESCRIPTION (provided by applicant): Ribosomes are large ribonucleoprotein complexes, which interact with protein factors and small RNA molecules to carry out the life-sustaining process of protein biosynthesis, or translation, in all cells, and in cellular organelles such as mitochondria. An understanding of structure and function of both bacterial and mitochondrial ribosomes (mitoribosomes) is essential in the effective treatment of bacterial infectious diseases. While bacterial ribosomes are the targets of several antibiotics, the mitoribosomes of the eukaryotic host cell must be protected from antibiotics if they are to continue the synthesis of crucial polypeptides required for the production of most of the cell's energy. Furthermore, defects in human mitochondrial translation are associated with several human genetic diseases. The long term goal of this study is to obtain structures of homologous functional complexes from bacterial and mitochondrial ribosomal systems, which will facilitate the identification of drug targets specific to the bacterial system; drugs can then be designed to inhibit bacterial translation without affecting the host's mitochondrial translation. We propose to study structures of functional complexes of both bacterial ribosomes and mammalian mitoribosomes by three-dimensional (3D) cryo-electron microscopy (cryo-EM). In the case of bacterial ribosomes, we will focus on complexes of elongation factor G (EF-G) and the ribosome recycling factor that undergo large-scale conformational changes during translation. We will label specific amino-acid residues of the protein factor with heavy-metal clusters and then visualize the label by 3D cryo-EM. These studies will allow us to characterize the dynamic behavior of the particular ligand that is essential for the functioning of the bacterial ribosome. Mammalian mitoribosomes are inherently poor candidates for crystallographic analysis, due to their compositional heterogeneity, and low abundance. We will determine cryo-EM structures for the mammalian mitoribosome complexed with mitochondrial initiation factor 2 and EF-G. The cryo-EM maps will be analyzed in terms of the atomic structures and homology models of the two types of ribosomes and their ligands, using various docking methods. This study will allow us to directly compare specific steps of translation, in bacterial and mammalian mitochondrial systems, and to identify potential drug targets. [unreadable] [unreadable] [unreadable]