Identification of a positive retroregulator that stabilizes mRNAs in bacteria.

A positive retroregulator that enhances the expression of an upstream gene(s) has been identified. It resides within a 381-base pair (bp) restriction fragment containing the transcriptional terminator of the crystal protein (cry) gene from Bacillus thuringiensis vs. Kurstaki HD-1. This fragment was fused to the distal ends of either the penicillinase (penP) gene of Bacillus licheniformis or the interleukin 2 cDNA from the human Jurkat cell line. In both cases, the half-lives of the mRNAs derived from the fusion genes were increased from approximately equal to 2 to 6 min in both Escherichia coli and Bacillus subtilis. Synthesis of the corresponding polypeptides in the bacteria carrying the fusion genes was also increased correspondingly. The enhancement of expression of the upstream genes was independent of the insertional orientation of the distal cry terminator fragment. Deletion analysis showed that the locus conferring the enhancing activity coincided with the terminator sequence and was located within a 89-bp fragment that includes an inverted repeat, the 19-bp upstream-, and the 27-bp downstream-flanking sequences. We propose that transcription of the retroregulator sequence leads to the incorporation of the corresponding stem-and-loop structure at the 3' end of the mRNA; the presence of this structure protects the mRNAs from exonucleolytic degradation from the 3' end and, thereby, increases the mRNA half-life and enhances protein synthesis of the target genes.