Overexpression of tnaC of Escherichia coli Inhibits Growth by Depleting tRNA 2 Pro 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 tRNA(2)(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-tRNA(2)(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 tRNA(2)(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 tRNA(1)(Pro), a tRNA(Pro) that translates CCG, but not CCU. Replacing the Pro24CCU codon of tnaC by Pro24CCG, a Pro codon translated by tRNA(1)(Pro), also led to growth rate reduction, and this reduction was relieved by overexpression of tRNA(1)(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.