The objective of this project is the investigation of structure-function relationships in the Drosophila ribosome, with emphasis on the mechanism and sites of binding of eukaryotic-specific, protein synthesis antibiotics. This work has three complementary lines of research: (1) use of previously characterized ribosome affinity labels to further map the peptidyl transferase site of Drosophila ribosomes, (2) the development and use of antibiotic-derived affinity labels, and (3) the isolation and characterization of antibiotic-resistant drosophila ribosomes derived from resistant cell lines. The development of new affinity labels will begin with the testing of five eukaryotic-specific protein synthesis inhibitors, anisomycin, trichodermin, cyclohexide, MDMP and emetine for their ability to be directly or photoinducibly reactive towards ribosomal components. Any antibiotics whch are found to be unreactive will be modified to contain a chemically reactive group and tested again for binding and covalent attachment to ribosomes. Simultaneously, we plan to mutagenize Schneider's line 2 cells and select for antibiotic-resistant clones. Resistant ribosomes will be investigated for any altered ribosomal components by electrophoresis and antibiotic binding studies as well as altered affinity labeling patterns. These results can be compared to produce a picture of those ribosomal components responsible for antibiotic sensitivity and themechanisms for the change to resistance. By comparing these data with other systems we may gain insight into the evolution of the protein synthesis machinery. The drug-resistant cell lines will be useful for genetic as well as biochemical studies on the gene expression of ribosomal components. But, most importantly, studies such as these can yield valuable information on the basic structure-function mechanisms of protein synthesis and eventually lead to the design of more specific, effective antibiotics.
NIH ExPorter
NIH ExPorter