The bacterial ribosome has become one of the better understood cellular organelles through the application of a variety of structural techniques. The complete primary structures of both RNA and protein components are known and approximate locations for many proteins and termini of the RNA have been established. Despite this abundance of information, the molecular mechanism of ribosome function remains elusive. Comparative structural evidence points to a major role for RNA in the primitive translational machinery. Ribosomal proteins clearly play a central role in the highly evolved ribosome. This proposal deals with the protein topography of ribosomes on several structural and functional levels. Crosslinking with bifunctional protein specific reagents will be used to elucidate specific peptides and residues at the contact sites between neighboring ribosomal proteins and between ribosomal proteins and bound elongation factors. Use will be made of the symmetrical crosslinking reagent 2-iminothiolane and of heterobifunctional crosslinking reagents that will be attached at specific residues in proteins of functional interest. Crosslinking at the translocation site will be used to investigate conformational changes associated with the cleavage of GTP. Crosslinking at the peptidyl transferase center, and at the translocation site, will be used to map at higher resolution the spatial arrangement of active site peptides which interact cooperatively in protein synthesis. Several monoclonal antibodies will be produced against active site proteins or peptides derived from them, and their sequential determinants will be identified. The antibodies will be used as tools for identifying crosslinked sites and as probes for analyzing the contribution of different parts of a protein to ribosome functions. They will also be used to map sites by immuno electron microscopy. The contribution of specific peptides and residues of active site proteins, to ribosome function will also be studied using in vitro mutagenesis of key proteins such as L2. Altered proteins isolated from overproducing strains carrying the cloned mutagenized gene will be characterized in vitro in assays of reconstituted ribosomes. The effect of the altered protein assembled into ribosomes in vivo will be studied by isolating temperature sensitive mutants from strains bearing the altered gene. The information will greatly increase the resolution of the map of the protein topography of ribosomal active sites and should lead to new insights into the mechanism of protein synthesis.
