The ribosome is the key component in the final and fundamental step of gene expression, protein synthesis. Comparative structural evidence points to a major role for RNA in the primitive translational machinery, but ribosome proteins clearly play an indispensable role in the function of the evolved ribosome. The focus of this research is on how specifi ribosomal proteins contribute to the structure and function of critical ribosome domains. Two of the domains to be studied are present on the large ribosomal subunits of E. coli:: 1) The factor binding domain is linked to the process of translocation and of which the essential component is the highly conserved element of ribosomal quaternary structure: the L10:(L7/L12)4 pentameric complex. The translocation factor, elongation factor-G, is a member of the G-protein superfamily fo which the ribosome acts as the GTPase activator. Its interaction with both L7/L12 in the large subunit and also with S12 in the small subunit will be investigated. 2) The peptidyl transferase domain of which a ke component is L2. A third domain is the regulatory region in the small subunits of eukaryotic ribosomes where phosphorylation of protein S6 stimulates protein synthesis. The work exploits the groundwork built up in the two past grant periods by applying now proven genetic, protein- engineering, modification, crosslinking and fluorescence methods to identify precisely the ribosomal location and interactions of critical domains and residues within the individual proteins. Work on the translocation domain will continue to define the mechanism of the highly conserved multi-copy L7/L12 protein, but extend to proteins L10, which binds the two L7/L12 dimers at nonequivalent sites, elongation factor G and S12. The protein variants to be constructed are of two types: thos with cysteine residues introduced at selected non-critical sites where they can be used directly for zero-length disulfide crosslinking, for photocrosslinking with heterobifunctional reagents, and for the attachment of SH specific probes either for high resolution electron microscopy (with D. G. Glitz, UCLA), or for fluorescence measurements (with D. Jameson, U Hawaii).
The aim i s to determine the interactions, and their dynamics, of the selected sites with other ribosomal component and with elongation factors. The second class of protein variants will be designed to alter or delete suspected critical regions of L2 and L7/L12 in order to identify functionally important residues or regions. The L2 variants will be tested in reconstituted ribosomes (with B. S. Cooperman, U. Penn). The regulatory region of eukaryotic protein S6 wil be modified by protein kinase-catalyzed thiophosphorylation in order to provide sulfhydryl sites for the attachment of extrinsic crosslinking an fluorescent probes to be used to map the region and its interactions wit initiation factors (with J. Hershey). The proposed research will lead to new insights into the roles of critical proteins in the mechanism of ribosome function in protein synthesis.
