Our overall goal is to obtain a detailed description of the E. coli ribosomal binding sites of antibiotics. Taken together with the known inhibitory effects of these antibiotics on ribosome function, and with the 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 and should also, in a more general way, contribute to a better understanding of the relationship between ribosome structure and function. Our approach is to irradiate radioactive antibiotics, or radioactive photolabile antibiotic derivatives, in the presence of ribosomes. If covalent incorporation meets the usual criteria for specific photoaffinity labeling, then determination of incorporation sites in both the protein and RNA moieties, and visualization of the antibiotic site by immunoelectron microscopy, provide the desired description of the ribosomal binding site. The current group of antibiotics being studied are the peptidyl transferase inhibitors, puromycin, lincomycin and blasticidin S, streptomycin, which at low concentrations induces misreading, and tetracycline, which inhibits aminoacyl tRNA binding. In addition, we plan to extend these studies to the class of antibiotics known as 2-deoxystreptamine amino-glycosides in the near future, in particular neomycin and paromomycin, which stimulate misreading and inhibit translocation. This group of inhibitors should allow us to obtain a detailed look at some of the important functional sites on the ribosome. This proposal contains a new plan for using rrn DNA to map labeled sites in ribosomal RNA. Also proposed are extensions of the application of immunoelectron microscopy to the study of photoaffinity-labeled ribosomes, and of our general approach to the study of chick embryo ribosomes.
NIH ExPorter
NIH ExPorter