Our overall goal in this work is to obtain a detailed description of the E. coli ribosomal binding sites of antibiotics and other ribosomal ligands of known function. Taken together with ongoing efforts in several laboratories directed toward obtaining a topographic map of the ribosome, such a description should provide the basis for a detailed understanding of the inhibitory effects of antibiotics on protein synthesis, as well as contribute to a better understanding of the relationship between ribosome structure and function. Such improved understanding could well lead to the development of improved antibiotics for therapeutic use. Our approach makes use of the techniques of affinity labeling, DNA.RNA hybridization, immunoelectron microscopy, reconstitution, and HPLC to characterize the sites and functional significance of antibiotic and rellated ligand binding to the ribosome. For the coming grant period, our immediate goals are: 1. To Map Protein at Sites of or Related to Antibiotic Binding. We will identify proteins into which antibiotic affinity labels incorporate and determine the specificity of such incorporation. Proteins prepared by HPLC will be used to reconstitute ribosomal subunits in which a single, affinity-labeled protein replaces its corresponding native protein and all other proteins are added as native proteins. Functional tests on such subunits should provide important evidence for placing particular proteins at an antibiotic binding site. Such subunits will also be used for immunoelectron microscopy experiments, allowing three-dimensional localization not only of the antibiotic but also of the protein into which it is incorporated. 2. Localize Site of Affinity Labeling of Ribosomal RNA. We will further improve our RNA DNA hybridization method for localizing sites of labeling in RNA, and extend such localization to the oligonucleotide level. 3. Synthesize and Test Other Antibiotic Affinity Labels. We plan a vigorous program of synthesis of radioactive affinity labels, both photolabile and electrophilic, to extend our work to antibiotics other than those (puromycin, tetracycline, and streptomycin) we have emphasized until now. These include: lincomycin, chloramphenicol, tiamulin, and tobramycin.
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