58] Competitive incorporation of inactive proteins into the ribosomal structure. A method to study ribosomal protein functions

Abstract

The chapter discusses the competitive incorporation of inactive proteins into the ribosomal structure, which is a method to study ribosomal protein functions. Localization of the proteins directly involved in the ribosomal activities is a necessary step in the study of the relationship between structure and function in the ribosome. A method that combines reconstitution of the particles and chemical modification of its individual components suggests that when a chemically inactivated protein is introduced into the ribosomal structure the functions involving that protein may be affected. For this purpose, particles disassembled by high-salt treatment can be reconstituted in the presence of one ribosomal protein previously inactivated by chemical modification. The reconstitution takes place in this case in the presence of the total complement of ribosomal components, and the modified protein competes with native molecules for the binding site in the reconstituted structure. The preferential incorporation of the modified or unmodified protein will be determined by its affinity for the binding site as well as by the relative concentration of the two species. The chemical modification of the protein is a critical step for the success of this technique. The alteration introduced in the molecule must affect its possible implication in the ribosomal functions without altering its affinity for the ribosomal structure too drastically for it to compete with the unmodified protein.