Tryptophan Regulation of the Escherichia coli Tryptophanase (TNA) Operon

Free L-tryptophan induces the expression of the Escherichia coli tna operon that specifies proteins necessary for catabolizing tryptophan. Regulation is effected by a transcriptional attenuation mechanism requiring translational arrest at the TnaC regulatory leader peptide in the 5? leader of the tna transcript. Interactions between the TnaC nascent regulatory peptide and the elements constituting the ribosomal peptide exit tunnel are implicated in the inhibition of the translating ribosome by free L-tryptophan.

In this study, genetic and biochemical analyses were used to investigate the role of specific residues of the TnaC peptide and of 23S rRNA regions that line the ribosomal exit tunnel in TnaC-mediated ribosome arrest. Highly conserved amino acids of TnaC and the 23S rRNA nucleotides predicted by structural models to interact with those TnaC residues were selected for analysis. TnaC residues Trp-12, Asp-16, and Ile-19 and 23S rRNA nucleotides A748-A752 as well as U2609 and A2058 are crucial for L-tryptophan-induced TnaC-mediated ribosome arrest. Interactions between the TnaC peptide and 23S rRNA residues are affected by mutations to either molecule. These interactions, specifically between Ile-19 of TnaC and the 23S rRNA A2058 nucleotide, are required for L-tryptophan binding and/or action. Finally, both cis-acting and trans-acting mutations can suppress the loss-of-function TnaC D16E mutation, supporting the model that both the TnaC peptide and the ribosome exit tunnel are active participants in the inhibition of peptidyl-transferase activity in response to L-tryptophan.

Taken together, the findings of this study suggest that the highly conserved nature of specific amino acids of TnaC can be explained by the requirement for interactions between these residues with 23S rRNA nucleotides within the ribosomal exit tunnel. These interactions likely induce conformational changes within the TnaC peptide, the ribosomal exit tunnel or both that contribute to the formation of a free L-tryptophan binding site, locking the peptidyl-transferase center in an inactive conformation resulting in ribosome arrest.