Multiple determinants of functional mRNA stability: sequence alterations at either end of the lacZ gene affect the rate of mRNA inactivation

Abstract

The Escherichia coli lacZ gene was used as a model system to identify specific sequence elements affecting mRNA stability. Various insertions and substitutions at the ribosome-binding site increased or decreased the rate of mRNA inactivation by up to fourfold. Deletion of a dyad symmetry, which may give rise to a very stable secondary structure in the mRNA immediately downstream of the gene, decreased the functional stability of the lacZ message. The magnitude of the latter effect was strongly dependent on the sequences at the ribosome-binding site, ranging from practically no effect for the most labile transcripts to a threefold decrease in stability for the most stable one. The results suggest that the wild-type lacZ message is inactivated predominantly by attacks near the ribosome-binding site, presumably in part because the putative secondary structure downstream of the gene protects against 3'-exonucleolytic attack. Taken together, the data for all of the modified variants of lacZ were shown to be quantitatively compatible with a general model of mRNA inactivation involving multiple independent target sites.