This project proposes to study the interaction of inhibitors of translation with the ribosome. Several experimental systems and research techniques will be used to investigate sites of antibiotic action. A collection of antibiotic resistant mutants of Halobacterium halobium will be used to study effects of mutations in rRNA on the binding and action of peptidyl-transferase targeted antibiotics. In particular, the pristinamycin-resistance mutation will be analyzed in order to better characterize the synergistic mode of action of streptogramin antibiotics. Cross-resistance effects conferred by individual antibiotic resistance mutations in domain V of 23S rRNA will be investigated. Mapping of the mutations responsible for resistance to various protein synthesis inhibitors in a number of selected mutants will be undertaken. The PI will gain more information about the structure of antibiotic binding sites in rRNA by footprinting with several peptidyl-transferase targeted antibiotics on the functionally active protein deficient particles prepared from the large ribosomal subunit of the thermophilic bacterium Thermus aquaticus. Accessibility of the extended rRNA regions in such particles should yield new information about molecular contacts between antibiotics and rRNA. Repeated selection-amplification cycles will be used for isolating rRNA fragments (or their complexes with ribosomal proteins) capable of interaction with antibiotics. Antibiotic-bound rRNA fragments will be isolated from a pool of random rRNA fragments. Functional activity of the isolated rRNA fragments and their interaction with the respective antibiotics will be studied. The PI will devote significant effort to understanding a novel mechanism of erythromycin resistance discovered recently in his laboratory. The resistance is mediated by overproduction in vivo of an rRNA-encoded short peptide which is capable of specific interaction with the ribosome. Other short peptides with similar properties will be isolated from expression libraries of random short peptides. Experiments with both in vitro transplanted and synthetic peptides will be used to understand structural requirements for peptide activity, as well as mechanism of peptide interaction with the ribosome. He will map regions of rRNA located in the vicinity of the nascent peptide exit channel; a probable binding site of erythromycin resistance peptides.
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