Overexpression of tnaC of Escherichia coli inhibits growth by depleting tRNA₂^Pro availability

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 tRNA ₂^Pro in the cell was sequestered in ribosomes as TnaC-tRNA₂^Pro. The half-life of this TnaC-tRNA ₂^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 tRNA ^Pro 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 tRNA^Pro depletion, resulting from TnaC-tRNA^Pro retention within stalled, translating ribosomes.