UC San Diego

In the termination phase of protein synthesis, class I release factors, RF1 and RF2, have the ability to recognize stop codons with great affinity and catalyze peptidyl-tRNA hydrolysis; however, the mechanism behind how this occurs is not well known. Recent crystal structures have allowed the investigation of critical residues in these release factors that may be critical to codon recognition as well as peptide release (Laurberg, et al. 2008). Therefore, the goal of this study is to investigate critical residues of RF1 and determine their role in the recognition process of codon specificity or in the hydrolysis of peptidyl-tRNA. To investigate the roles of Thr 186, Arg 182, Thr 194, and Gln 181, a recently developed fluorescence based assay was used to study the equilibrium binding of these mutants to the ribosome (Hetrick, et al. 2009). According to the results as seen by the change in fluorescence intensity, these mutants all seemed to have a major defect in binding. The next step was to check if these residues were crucial to peptide release; therefore, to eliminate the binding deficiency, saturated amounts of the factors were added to the ribosome complex, and the rate of catalysis was quantified through the peptide release assay. Surprisingly, all of the mutants were found to have similar rates of peptidyl- tRNA hydrolysis when compared to wild type RF1. Therefore, these mutants seem to be only involved in the recognition of the stop codons in the decoding center