Our project goal is to elucidate the mechanism of ribosome self-assembly. Two different approaches are being used to reach this objective. First, we are using a newly discovered inhibitor of ribosome assembly. This inhibitor, a dye called Cibacron Blue F3GA, can effectively stop assembly at any point in time without damage to the particle that has assembled up to that stage. This allows us to determine in detail the actual kinetic pathway of ribosome assembly. Furthermore, we should be able to determine the effect of various parameters such as temperature on the kinetics of assembly of each protein. This work is being carried out on both the 30s and the 50s ribosomal subunits. Our second approach involves the production of various fragments of individual proteins. We have used both enzymatic and chemical methods of cleavage. In addition, we have available a number of mutant strains of E. coli which are comprised of ribosomes carrying one of the proteins in a fragmented form. We have used a number of these selected fragments in the assembly reaction in place of the parent protein. The effect of the missing section on the mechanism of assembly and ribosome function is then assessed.
