Origins of minigene-dependent growth inhibition in bacterial cells

The expression of very short open reading frames in Escherichia coli can le ad to the inhibition of translation and an arrest in cell growth. Inhibitio n occurs because peptidyl-tRNA hydrolase fails to recycle sufficiently rapi dly peptidyl-tRNA released from ribosomes at the stop signal in competition with normal termination, causing starvation for essential species of tRNA. Previous studies have shown that the last sense codon, the strength of the Shine-Dalgarno sequence and the nature and context of the stop codon affec t the toxicity associated with mini-gene expression. Here, several importan t parameters are studied as a function of the length of the mini-gene codin g sequence. The rate of peptidyl-tRNA drop-off catalysed by translation fac tors decreases dramatically for peptides longer than a hexamer, The probabi lity that ribosomes recycle without dissociation of the mini-gene mRNA vari es strongly with the length of the coding sequence. The peptidyl-tRNA hydro lase rap mutant, unlike the wild-type enzyme, is highly sensitive to the le ngth and sequence of the peptide. Together, these parameters explain the le ngth dependence of minigene toxicity.