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Mol Microbiol. 2001 Jan;39(2):361-9.

Increased bar minigene mRNA stability during cell growth inhibition.

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Departamento de Biotecnología y Bioingeniería, CINVESTAV-IPN, Mexico City, Mexico.


Bacteriophage lambda is unable to grow vegetatively on Escherichia coli mutants defective in peptidyl-tRNA hydrolase (Pth) activity. Mutations which allow 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 different Shine-Dalgarno (SD) and spacer regions. The induced expression of barI+ and barII+ regions from plasmid constructs resulted in similar patterns of protein synthesis inhibition and cell growth arrest. Therefore, these deleterious effects may stem from translation of the transcripts containing the minigene two-codon 'ORF' (open reading frame). To test for this possibility, we assayed the effect of point mutations within the barI minigene. The results showed that a base pair substitution within the SD and the two-codon 'ORF' sequences affected protein synthesis and cell growth inhibition. In addition, mRNA stability was altered in each mutant. Higher mRNA stability 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 result of deficiency of specific tRNA.

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