Isolation of a mRNA instability sequence that is cis-dominant to the ompA stability determinant in Escherichia coli

Transcriptional fusions with ompA and bla have been used to identify a novel mRNA instability element. A 287-nucleotide (nt) sequence containing a repetitive extragenic palindrome (REP) from the chloramphenicol acetyltransferase (cat) gene was inserted into the 3' untranslated region (UTR) of the ompA gene. In one orientation, the insert had no effect on the half-life of the ompA-cat chimeric transcript. In the other orientation, however, the sequence functioned as a destabilizing element and was dominant to the 5'-UTR ompA and REP stability elements. The orientation-dependent effect of the instability sequence suggests that sequence rather than structure alone is important to the function of the instability determinant. In addition, the instability sequence also destabilized an ompA-bla fusion construct when fused to its 3'-UTR region. A sensitive RNA ligation/PCR amplification technique was developed and used to analyze RNA decay intermediates. The results indicated that degradation of the chimeric transcript initiated within the 287-nt inserted sequence.