Stability of the Osmoregulated Promoter-DerivedproPmRNA Is Posttranscriptionally Regulated by RNase III in Escherichia coli

Abstract

ABSTRACTThe enzymatic activity ofEscherichia coliendo-RNase III determines the stability of a subgroup of mRNA species, includingbdm,betT, andproU, whose protein products are associated with the cellular response to osmotic stress. Here, we report that the stability ofproPmRNA, which encodes a transporter of osmoprotectants, is controlled by RNase III in response to osmotic stress. We observed that steady-state levels ofproPmRNA and ProP protein are inversely correlated with cellular RNase III activity and, in turn, affect the proline uptake capacity of the cell.In vitroandin vivoanalyses ofproPmRNA revealed RNase III cleavage sites in a stem-loop within the 5′ untranslated region present only inproPmRNA species synthesized from the osmoregulated P1 promoter. Introduction of nucleotide substitutions in the cleavage site identified inhibited the ribonucleolytic activity of RNase III onproPmRNA, increasing the steady-state levels and half-life of the mRNA. In addition, decreased RNase III activity coincided with a significant increase in both the half-life and abundance ofproPmRNA under hyperosmotic stress conditions. Analysis of the RNA bound to RNase III viain vivocross-linking and immunoprecipitation indicated that this phenomenon is related to the decreased RNA binding capacity of RNase III. Our findings suggest the existence of an RNase III-mediated osmoregulatory network that rapidly balances the expression levels of factors associated with the cellular response to osmotic stress inE. coli.IMPORTANCEOur results demonstrate that RNase III activity onproPmRNA degradation is downregulated inEscherichia colicells under osmotic stress. In addition, we show that the downregulation of RNase III activity is associated with decreased RNA binding capacity of RNase III under hyperosmotic conditions. In particular, our findings demonstrate a link between osmotic stress and RNase III activity, underscoring the growing importance of posttranscriptional regulation in modulating rapid physiological adjustment to environmental changes.