Expression of peptide chain release factor 2 requires high-efficiency frameshift

Abstract

Peptide chain release factors are soluble proteins that participate in the stop codon-dependent termination of polypeptide biosynthesis. In Escherichia coli, two release factors are necessary for peptide chain termination: release factor 1 (RF1) specifies UAG-and UAA-dependent termination whereas release factor 2 (RF2) specifies UGA- and UAA-dependent termination¹. Release factors are found in low concentrations relative to other translation factors², suggesting that their expression is tightly regulated and, accordingly, making the study of their structure–function relationship difficult. RF1 and RF2 exhibit significant sequence homology, probably reflecting their similar functions and perhaps a common evolutionary origin³. DNA and peptide sequencing have suggested the existence of a unique mechanism for the autogenous regulation of RF2 in which an in-frame UGA stop codon requires an obligatory +1 frameshift within the coding region of the RF2 gene. In this report we present in vitro experimental results consistent with the autogenous regulation of RF2. Additionally, we used RF2-lacZ gene fusions to demonstrate that autogenous regulation occurs, at least in part, by premature termination at the in-frame stop codon, since deletion of this stop codon leads to overproduction of the RF2–LacZ fusion protein. Frameshifting at this premature termination codon occurs at the remarkably high rate of 50%.