The ribosome is the unique site of protein biosynthesis in all cells, and as such a detailed understanding of its structure and function is of fundamental importance to the more general understanding of cellular function at the molecular level. Our studies will be carried out ont he E. coli ribosome, which is by far the best characterized by the studies of many groups, including our own. However, given the considerable conservation of ribosome structure throughout evolution, the results we obtain should also be useful for understanding ribosomes from other organisms. We will continue our use of radioactive, photolabile derivatives of oligo DNAs having sequence complementary to single-stranded rRNA sequences for this purpose. Such probes can bind to their targeted sequences in intact ribosomal subunits, and, on photolysis, incorporate into neighboring ribosomal components that can subsequently be identified by methods perfected in our laboratory. We will expand the scope of this work by: varying the size of the spacer linking the photolabile group to the complementary base; varying the site of attachment of the photolabile group within the oligoDNA probe; and using photolabile oligoDNA probes to monitor conformational change in the vicinity of the target rRNA sequence. We also will explore the utilization of a second approach based on the replacement of phosphate with thiophosphate or U with 4-thioU at specific locations within rRNA. The electrophilic sulfurs thus introduced into RNA provide specific sites for the incorporation of photolability (of course, the 4-thioU is itself photolabile). Reconstitution of ribosomes with such modified rRNA, followed by photolysis, will allow neighboring components to be identified for rRNA sites that are inaccessible to oligoDNA probes. The proposed studies will provide information critical for the construction of the three-dimensional structure of the ribosome, a requirement for understanding ribosomal function. Aside from its intrinsic importance to the basic comprehension of life processes, better understanding of ribosomal function could have important therapeutic consequences. Many antibiotics in current clinical use, such as tetracycline, erythromycin and other macrolides, neomycin and other aminoglycosides, and chloramphenicol target ribosomes as their sites of action. Interest in these ribosomal antibiotics has been growing s bacterial resistance to beta-lactams and quinolines has become more widespread. Several drug companies are now devoting considerable resources toward synthesizing analogues and derivatives of ribosomal antibiotics that overcome bacterial resistance. Better understanding of ribosomal structure and function will be especially important for antibiotics, such as macrolides, where resistance is based on changes in ribosome structure.