The fundamental importance of the ribosome in cellular function is evidenced by its universality and fundamental conservation throughout all forms of life. Functional complexes of the bacterial ribosome undergo large conformational changes during the course of protein synthesis. Recent dramatic progress in the elucidation of ribosome structure by both X-ray crystallography and cryoelectron microscopy has provided some of the best evidence for such changes. At the same time, detailed rate studies of initiation and elongation partial reactions, even though for the most part incomplete, have shown each of these processes to be complex, multistep reactions, raising the question of the extent to which specific structural changes can be assigned to specific steps described in proposed kinetic mechanisms. Our work has the overall goals of a) fully elucidating the kinetic mechanisms of initiation and elongation for wild-type ribosomes using fluorescence stopped-flow, quenched flow, and FRET approaches, focusing on three G- protein factors, EF-G, EF-Tu, and IF2, and the ribosomal site with which they all interact, the so-called GAC (GTPase Activation Center; and b) determining the importance of specific interactions for specific steps of ribosome catalysis by conducting structure-function analyses, measuring how specific perturbations in the protein synthesis machinery affect rates of specific steps of protein synthesis. The specific perturbations will be induced by mutation of tRNA, mutation of the peptidyl transferase center (PTC), and by addition of antibiotics targeting either the GAC (thiostrepton) or the PTC (oxazolidinones and sparsomycin). The improved understanding of ribosomal function that will result from our work could have important consequences for the design, testing, and utilization of new ribosomal antibiotics, since it should provide a paradigm for determining the specific effect(s) of a given antibiotic on ribosomal function, which can be linked directly to knowledge of the site of antibiotic binding. Medical interest in ribosomal antibiotics has been growing as bacterial resistance to beta-lactams and quinolines has become more widespread. Indeed, major pharmaceutical companies are now devoting considerable resources toward both the screening and rational design approaches for the discovery of new ribosomal antibiotics, as well as toward the modification of known antibiotics so as to overcome bacterial resistance while retaining strong inhibitory activity, largely as a result of the successful development of the new oxazolidinone family of ribosomal antibiotics and of the increased knowledge of the structure and function of the bacterial ribosome.
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