Search for: All records

Abstract Free L-tryptophan (L-Trp) stalls ribosomes engaged in the synthesis of TnaC, a leader peptide controlling the expression of the Escherichia coli tryptophanase operon. Despite extensive characterization, the molecular mechanism underlying the recognition and response to L-Trp by the TnaC-ribosome complex remains unknown. Here, we use a combined biochemical and structural approach to characterize a TnaC variant (R23F) with greatly enhanced sensitivity for L-Trp. We show that the TnaC–ribosome complex captures a single L-Trp molecule to undergo termination arrest and that nascent TnaC prevents the catalytic GGQ loop of release factor 2 from adopting an active conformation at the peptidyl transferase center. Importantly, the L-Trp binding site is not altered by the R23F mutation, suggesting that the relative rates of L-Trp binding and peptidyl-tRNA cleavage determine the tryptophan sensitivity of each variant. Thus, our study reveals a strategy whereby a nascent peptide assists the ribosome in detecting a small metabolite. 

The tnaC regulatory gene from the tna operon of Escherichia coli 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 E. coli translation termination factors, RF1 and RF2. Using a series of reporter genes, we found that the in vivo L-Trp sensitivity of tnaC gene expression is influenced by the identity of its stop codon, with the UGA stop codon producing higher expression efficiency of the tnaA-lacZ gene construct than the UAG stop codon. in vitro TnaC-peptidyl-tRNA accumulation and toeprinting 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 tnaC(UGA) tnaA-lacZ 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 tnaC gene. This inhibition depends on the identities of amino acid residues surrounding the RF2-GGQ functional motif.