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. 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 as 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. Our studies will be carried out on the 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. The overall goal of this proposal is to describe conformational changes that the ribosome undergoes during specific steps of its functional cycle and how mutations and antibiotic binding affect these changes. We propose to do this by forming defined photocrosslinks from rRNA sites within the ribosome that have been targeted on the basis of their importance for ribosome structure and function, taking advantage of the intrinsic ability of the photocrosslinking process to sample all conformations in solution. Such crosslinks will be formed in different functional states, in wild-type and mutant ribosomes, and in the presence and absence of antibiotics. The structural constraints represented by such crosslinks, along with constraints generated by other approaches, will be used to model structures of the ribosome in specific functional states, using crystal structures of 70S ribosomes and 30S and SOS subunits as initial structures. As our major approach we will continue and refine the use of radioactive, photolabile derivatives of oligonucleotides having sequences complementary to rRNA sequences (PHONTs). Such probes bind to their targeted sequences in intact ribosomal subunits, and, on photolysis, incorporate into neighboring ribosomal components that can subsequently be identified. We also will develop a second approach based on site-specific introduction of photolability into intact rRNA (IPHOR - intact photolabile RNA) to obtain similar information for rRNA sites that are either inaccessible to PHONTs or where the use of PHONTs induces major conformational change.
