Increased bar minigene mRNA stability during cell growth inhibition

Bacteriophage lambda is unable to grow vegetatively on Escherichia coli mut ants defective in peptidyl-tRNA hydrolase (Pth) activity. Mutations which a llow phage growth on the defective host have been located at regions named bar in the lambda genome. Expression of wild-type bar regions from plasmid constructs results in inhibition of protein synthesis and lethality to Pth- defective cells. Two of these wild-type bar regions, barI(+) and barII(+), contain minigenes with similar AUG-AUA-stop codon sequences preceded by dif ferent Shine-Dalgarno (SD) and spacer regions. The induced expression of ba rI(+) and barII(+) regions from plasmid constructs resulted in similar patt erns of protein synthesis inhibition and cell growth arrest. Therefore, the se deleterious effects may stem from translation of the transcripts contain ing the minigene two-codon 'ORF' (open reading frame). To test for this pos sibility, we assayed the effect of point mutations within the barI minigene . The results showed that a base pair substitution within the SD and the tw o-codon 'ORF' sequences affected protein synthesis and cell growth inhibiti on. In addition, mRNA stability was altered in each mutant. Higher mRNA sta bility correlated with the more toxic minigenes. We argue that this effect may be caused by ribosome protection of the mRNA in paused complexes as a r esult of deficiency of specific tRNA.