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. The long term goal of work in this laboratory is to create a detailed structure-function map of the ribosome. The work put forward in this proposal is an important part of this effort, since it provides a uniquely powerful approach for obtaining a detailed picture of the ribosomal components that neighbor specific sequences of rRNA within intact ribosomes and subunits. Specifically, The PI proposes to exploit radioactive, photolabile, complementary oligoDNA probes of ribosomal RNA (rRNA) as photoaffinity labels to identify ribosomal components that neighbor targeted rRNA sequences within an intact ribosome or ribosomal subunit and to determine how the identities of such components vary in different functional sites of the ribosome. The secondary structures of rRNA are well established. The targeted sequences fall in single-stranded regions having particular functional or structural significance, including those known or suspected to be involved in the peptidyl transferase center, in the anticodon-codon recognition center, in 30S:50S subunit contacts, and in the binding of specific ribosomal ligands such as protein factors that stimulate protein synthesis (e.g.,EF- G,IF3) and of antibiotics. These studies will be carried out on the E. coli ribosome, which is by far the best characterized. However, given the considerable conservation of ribosome structure throughout evolution, the results obtained should also be useful for understanding ribosomes from other organisms.
