RIBOSOMAL FUNCTION & ANTIBIOTIC DESIGN

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DESCRIPTION (provided by applicant): The recent availability of several x-ray crystal structures for the ribosome have created considerable excitement in the field. These structures will be instrumental in devising experiments to elucidate the details of ribosomal synthesis of proteins. Furthermore, the bacterial ribosome is the target of many classes of antibiotics, each of which interferes with an aspect of the biochemistry of protein synthesis. Studies of ribosome structures will lead the way in developing novel classes of antibiotics in the near future. The long-term goal of this multidisciplinary collaborative project is to utilize the structural information from known x-ray structures to study ribosome function in greater detail and to develop novel inhibitors of an essential and universal biosynthetic process. Four specific aims are proposed. 1) A combination of molecular dynamics simulations and experiments will be used to explore the dynamic nature of the decoding process during peptide-bond formation and to understand the role of antibiotic binding in this process. 2) Two molecules that have been designed to explore the motion of the rRNA A site during the decoding process will be synthesized and studied for rRNA binding. These compounds are potential inhibitors of A site function, hence possible antibacterials. 3) Two iron complexes of aminoglycoside-EDTA complexes will be synthesized and tested for their ability to bind the ribosome and fragment the rRNA backbone at sites located within the antibiotic-binding site(s). These experiments will identify the locations in which these antibiotics bind, as well as provide information about dynamics through examination of cleavage patterns. 4) Specific aim 4 expands on earlier success in the design of new antibiotics. A series of molecules that will interfere with protein synthesis by binding to the ribosomal A site will be generated. A host of specific analyses (assessment of antibacterial properties with living bacteria, translation assays, bacterial membrane permeability assay, assays for DNA and RNA function in bacteria, cytotoxicity assays, assays with A-site mutated 30S ribosomes and bacteria that harbor them, and x-ray analyses of the 30S ribosome and the A-site model complexed with the novel antibiotics) have been envisioned for the study of the antibacterial properties of these molecules. This collective effort will shed light both on the various molecular events within the ribosomal and on how to inhibit some of these processes.