Molecular contributions to catalysis during translation termination on the ribosome

The active site of the large ribosomal subunit is composed entirely of RNA and catalyzes the two related reactions of peptide bond formation and peptide release. The more chemically challenging release reaction uses a water nucleophile and is catalyzed by class I release factor protein in response to stop codon recognition in the A site of the small subunit. The release factor protein contains a universally conserved GGQ motif that interacts with the active site and is critical for peptide release, although its role in the reaction is not well defined. Testing the specificity of the release reaction with alternate nucleophiles, we found that release factor contributes to a general activation of the ribosome active site, consistent with an induced-fit mechanism previously implicated in peptide bond formation. We also found that mutations in the conserved GGQ glutamine of RF1 can affect the specificity of the release reaction, indicating that this amino acid is in close proximity with the reaction nucleophile.;The 2' OH of the peptidyl-tRNA substrate was known to be critical for peptide bond formation and believed to participate in a proton shuttle mechanism in the ribosomal active site. Our studies also found this moiety to be essential for catalysis of peptide release. Deoxy substitutions in the tRNA substrate affected its activity in the uncatalyzed reaction on the ribosome, but not free in solution indicating that the 2' OH is only important in the context of the ribosomal active site. Subsequent work identified mutants in the RF1 GGQ glutamine that can rescue defects associated with loss of the peptidyl-tRNA substrate 2' OH. These results suggest that the P-site substrate 2' OH is critical for the reaction on the ribosome because it is the only group available to shuttle a proton from the nucleophile to the 3' OH leaving group. The work presented in this thesis represents significant progress in identifying the role of molecular components that are essential for peptide release catalysis.