Reduced action of polypeptide release factors induces mRNA cleavage and tmRNA tagging at stop codons in Escherichia coli

Abstract

Certain C-terminal sequences of nascent peptide cause an efficient protein tagging by tmRNA system at stop codons in Escherichia coli. Here, we demonstrate that both mRNA cleavage and tmRNA tagging occur at UAG stop codon recognized specifically by polypeptide release factor 1 (RF-1) when the activity of RF-1 is reduced by a mutation in the prfA gene without requirement of particular C-terminal sequences of nascent peptide. The tmRNA tagging and mRNA cleavage in the prfA mutant were eliminated when the wild-type RF-1 but not RF-2 was supplied from plasmid. In addition, depletion of either RF-1 or RF-2 induces endonucleolytic cleavage and tmRNA tagging at UAG or UGA stop codons respectively. We conclude that ribosome stalling at the cognate stop codon caused by reduced activity or expression of RF-1 or RF-2 is responsible for mRNA cleavage. The present data along with our previous studies strongly suggest that ribosome stalling leads to endonucleolytic cleavage of mRNA in general resulting in non-stop mRNA and that the 3' end of non-stop mRNA is probably only target for the tmRNA system.