The regulatory TnaC nascent peptide preferentially inhibits release factor 2-mediated hydrolysis of peptidyl-tRNA

Abstract

The <i>tnaC</i> regulatory gene from the <i>tna</i> operon of <i>Escherichia coli</i> controls the transcription of its own operon through an attenuation mechanism relying on the accumulation of arrested ribosomes during inhibition of its own translation termination. This free l-Trp-dependent mechanism of inhibition of translation termination remains unclear. Here, we analyzed the inhibitory effects of l-Trp on the function of two known <i>E. coli</i> translation termination factors, RF1 and RF2. Using a series of reporter genes, we found that the <i>in vivo</i> l-Trp sensitivity of <i>tnaC</i> gene expression is influenced by the identity of its stop codon, with the UGA stop codon producing higher expression efficiency of the <i>tnaA-lacZ</i> gene construct than the UAG stop codon. <i>In vitro</i> TnaC-peptidyl-tRNA accumulation and toe-printing assays confirmed that in the presence of l-Trp, the UGA stop codon generates higher accumulation of both TnaC-peptidyl-tRNA and arrested ribosomes than does the UAG stop codon. RF-mediated hydrolysis assays corroborated that l-Trp blocks RF2 function more than that of RF1. Mutational analyses disclosed that amino acids substitutions at the 246 and 256 residue positions surrounding the RF2-GGQ functional motif reduce l-Trp-dependent expression of the <i>tnaC(UGA) tnaA-lacZ</i> construct and the ability of l-Trp to inhibit RF2-mediated cleavage of the TnaC-peptidyl-tRNA. Altogether, our results indicate that l-Trp preferentially blocks RF2 activity during translation termination of the <i>tnaC</i> gene. This inhibition depends on the identities of amino acid residues surrounding the RF2-GGQ functional motif.