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

Stop codon recognition is a crucial event during translation termination and is performed by class I release factors (RF1 and RF2 in bacterial cells). Recent crystal structures showed that stop codon recognition is achieved mainly through a network of hydrogen bonds and stacking interactions between the stop codon and conserved residues in domain II of RF1/RF2. Additionally, previous studies suggested that recognition of stop codons is coupled to proper positioning of RF1 on the ribosome, which is essential for triggering peptide release. In this study we mutated four conserved residues in Escherichia coli RF1 (Gln185, Arg186, Thr190, and Thr198) that are proposed to be critical for discriminating stop codons from sense codons. Our thermodynamic and kinetic analysis of these RF1 mutants showed that the mutations inhibited the binding of RF1 to the ribosome. However, the mutations in RF1 did not affect the rate of peptide release, showing that imperfect recognition of the stop codon does not affect the proper positioning of RF1 on the ribosome.