Overexpression of tnaC of Escherichia coli Inhibits Growth by Depleting tRNA2Pro Availability

ABSTRACT Transcription of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. Induction results from ribosome stalling after translation of tnaC, the coding region for a 24-residue leader peptide. The last sense codon of tnaC, proline codon 24 (CCU), is translated by tRNA2 Pro. We analyzed the consequences of overexpression of tnaC from a multicopy plasmid and observed that under inducing conditions more than 60% of the tRNA2 Pro in the cell was sequestered in ribosomes as TnaC-tRNA2 Pro. The half-life of this TnaC-tRNA2 Pro was shown to be 10 to 15 min under these conditions. Plasmid-mediated overexpression of tnaC, under inducing conditions, reduced cell growth rate appreciably. Increasing the tRNA2 Pro level relieved this growth inhibition, suggesting that depletion of this tRNA was primarily responsible for the growth rate reduction. Growth inhibition was not relieved by overexpression of tRNA1 Pro, a tRNAPro that translates CCG, but not CCU. Replacing the Pro24CCU codon of tnaC by Pro24CCG, a Pro codon translated by tRNA1 Pro, also led to growth rate reduction, and this reduction was relieved by overexpression of tRNA1 Pro. These findings establish that the growth inhibition caused by tnaC overexpression during induction by tryptophan is primarily a consequence of tRNAPro depletion, resulting from TnaC-tRNAPro retention within stalled, translating ribosomes.