Function of polypeptide chain release factor RF-3 in Escherichia coli. RF-3 action in termination is predominantly at UGA-containing stop signals

Abstract

Two protein release factors (RFs) showing codon specificity, RF-1 (UAG, UAA) and RF-2 (UAA, UGA), are required for polypeptide chain termination in Escherichia coli. We recently reported the localization and characterization of the gene encoding RF-3 (prfC), a third protein component previously described as stimulating termination without codon specificity. RF-3 is a GTP-binding protein that displays much sequence similarity to elongation factor EF-G. In a termination assay in vitro, RF-3 lowers the Km for terminator trinucleotides and is thought to act in termination signal recognition. The gene prfC was identified by transposon insertion mutagenesis leading to enhanced nonsense suppression of UGA. We report here that (i) RF-3 inactivation significantly enhances the suppression of termination in vivo only at UGA-dependent stop signals; (ii) the codon-dependent contribution to the stimulation of fMet release in vitro by RF-3 is significantly greater with UGA termination triplet than UAG termination triplet; (iii) RF-3 increases dramatically the affinity of RF-2 to the UGA termination complex in vitro but not that of RF-1 to the UAG termination complex; (iv) RF-3 inactivation leads to a positive feedback on the autoregulation of RF-2 synthesis in vivo, dependent on the competition between frameshifting and termination. These findings are discussed in terms of the mechanism of involvement of RF-3 in translation termination.