Messenger RNA-directed protein synthesis is catalyzed in all cells by a highly conserved ribonucleoprotein enzyme called the ribosome. Atomic resolution crystal structures of ribosomes, the first of which were reported 4 years ago, have had a revolutionary impact on our understanding of protein synthesis, but many interesting questions remain that can best be approached crystallographically. The research proposed for the next 5 years has two components. First, we intend to use the crystallographic and genetic tools already developed here for Haloarcula marismortui (Hma) to obtain new insights into ribosome structure and function. Second, crystals of ribosomes and ribosomal subunits will be prepared from new species so that questions about ribosome structure and function can be answered that cannot be addressed using any of the ribosome crystals now available. Specifically, the hypothesis that the well known differences in the properties of the peptidyl transferase centers of the ribosomes from different species are caused by interactions between the nucleotides in its conserved core with more remote, non-conserved nucleotides will be tested in the Hma large ribosomal subunit using a combination of genetics and crystallography. The same tools will also be used to determine how the peptidyl transferase center responds conformationally to the mutation of highly conserved bases in the peptidyl transferase center. In addition, a series of experiments will be carried out the objective of which is to prepare large subunit that have nascent peptides in their exit tunnels, and solve their structures crystallographically. The final goal is the preparation of crystals of eukaryotic ribosomes that diffract to atomic resolution, and the determination of their structures.
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