RNase III Controls the Degradation ofcorAmRNA in Escherichia coli

Abstract

cIn Escherichia coli, the corA gene encodes a transporter that mediates the influx of Co 2؉ , Mg 2؉, and Ni 2؉ into the cell. During the course of experiments aimed at identifying RNase III-dependent genes in E. coli, we observed that steady-state levels of corA mRNA as well as the degree of cobalt influx into the cell were dependent on cellular concentrations of RNase III. In addition, changes in corA expression levels by different cellular concentrations of RNase III were closely correlated with degrees of resistance of E. coli cells to Co 2؉ and Ni 2؉. In vitro and in vivo cleavage analyses of corA mRNA identified RNase III cleavage sites in the 5=-untranslated region of the corA mRNA. The introduction of nucleotide substitutions at the identified RNase III cleavage sites abolished RNase III cleavage activity on corA mRNA and resulted in prolonged half-lives of the mRNA, which demonstrates that RNase III cleavage constitutes a rate-determining step for corA mRNA degradation. These findings reveal an RNase III-mediated regulatory pathway that functions to modulate corA expression and, in turn, the influx of metal ions transported by CorA in E. coli.

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

“…The predicted secondary structure of the hairpin was confirmed by analyzing the cleavage patterns of the model hairpin RNA after RNase T1 digestion. The other minor cleavage products observed might have resulted from the intrinsic ability of RNase III to randomly cleave RNA transcripts at high RNase III concentrations (8)(9)(10). We also synthesized a 3=-end 32 P-labeled model hairpin and performed an in vitro cleavage assay.…”

“…Genome-wide analyses of Escherichia coli transcripts indicated that the abundance of a large number of mRNA species is regulated by RNase III (4,5). The abundance of several E. coli mRNA transcripts is dependent on the endoribonucleolytic activity of RNase III, including rnc (6), pnp (7), bdm (8), betT (9), corA (10), proU (11), mltD (12), and rng (4). Studies indicate that a subgroup of mRNA transcripts encoding factors associated with the cellular response of E. coli to osmotic stress is regulated by RNase III (8,9,11).…”

“…Northern blot analysis. The procedure for Northern blot analysis has been described previously (10). The random hexamer probes used for proP mRNA detection were synthesized with a random primed DNA labeling kit (Roche, Pleasanton, CA).…”

“…His-tagged RNase III purification and cleavage assays were performed as previously described (31,32). The procedure used to synthesize 3=-or 5=-end-labeled transcripts was previously described (10). Primers T7-proP-F (5=-TAATACGACTCACTATAGGG TCATTAACTGCCCAATTCA-3=) and T7-proP-R (5=-TTATTCATCAA TTCGCGGAT-3=) were used to synthesize the full-length proP mRNA transcript, and Prop-st-T7-F (5=-CTTAATACGACTCACTATAGGGCA GGCGTCAACTGGTTTGATTGTACATTCC-3=) and Prop-st-T7-R were used to synthesize the model hairpin transcript.…”

“…The procedure used for primer extension analysis has been described previously (8,10). The primer used was prop-125R (5=-CCGCAAAAGGGGAGCGGGAA-3=).…”