Ribosomes, the cellular organelles that function as the factories for protein synthesis, are composed of several RNAs and 50 or more proteins. Although several key functions of the ribosome are catalyzed by the RNA, ribosomal proteins probably also play a key role in ribosome function. Such roles might include regulation of the activity of the RNA-based peptidyl transferase, control of the migration of nascent protein through the ribosome exit tunnel, and guidance of the peptide chain into the chaperone system that assists proper folding of nascent proteins. The Zengel laboratory has developed methods for in vivo synthesis of 50S ribosomal subunits containing mutant ribosomal protein L4 or L22. The activity of such mutant ribosomes will be analyzed in order to determine which ribosomal functions are dependent on L4 and L22. The work will focus particularly on versions of L4 or L22 lacking the characteristic tentacle that extends from the globular surface domain to the peptide exit tunnel. Preliminary experiments indicate that tentacle-less L4 or L22 can be stably incorporated into subunits that pair with 30S subunits and enter polysomes. Ribosomes containing mutant L4 or L22 will be analyzed for their in vivo and in vitro translation activity. This project will contribute to the elucidation of the molecular mechanisms of ribosome function, an essential function for all cells in all forms of life, and will provide training for the next generation of biologists, both at the undergraduate and graduate level and including significant numbers of minority students.