The ribosome is the large (2.5 MDa) cellular machine responsible for the synthesis of proteins. During decoding of the genetic message, this complex macromolecular complex selects the correct tRNA with both speed and accuracy. The recent high resolution X-ray and cryo-EM structures of the ribosome have shown that this selection process involves a myriad of ribosome-tRNA interactions. Moreover, the selection processes also trigger structural rearrangements that result in the closure of ribosomal domains around the correct tRNA. The functional significance of most of these tRNA-ribosome interactions and associated structural rearrangements is unknown. This research project will use genetic techniques to identify the critical ribosomal features and ribosome-tRNA contacts that are required for accurate protein synthesis. Selection schemes have been designed that will allow the recovery of ribosomal mutants with altered decoding fidelity. These mutants will be further characterized in the laboratory using a series of in vivo assays. In addition, collaborations have been established that will allow biochemical and structural characterization of the mutant ribosomes. Together, these combined approaches will lead to a clearer understanding of the decoding step of protein synthesis. Since decoding is a target for many currently-used antibiotics, a deeper understanding of this process may aid the development of new antibacterial agents and strategies to combat resistance. The project involves the training of post-doctoral, undergraduate and graduate students in modern molecular biology, as well as in classical bacterial and molecular genetics.
