RNase III autoregulation: structure and function of rncO, the posttranscriptional "operator"

Expression of the Escherichia coli rnc-era-recO operon is regulated posttranscriptionally by ribonuclease III (RNase III), encoded in the rnc gene. RNase III initiates rapid decay of the rnc operon mRNA by cleaving a double-stranded region of the rnc leader. This region, termed rncO, is portable, conferring stability and RNase III regulation to heterologous RNAs. Here, we report the detailed analysis of rncO structure and function. The first 215 nt of the rnc leader are sufficient for its function. Dimethylsulfate (DMS) modification in vivo revealed distinct structural elements in this region: a 13-nt single-stranded 5' leader, followed by a 6-bp stem-loop structure (I), a larger stem-loop structure (II) containing the RNase III site, a single-stranded region containing the rnc translation initiation site, and a small stem-loop structure (III) at the 3' terminus of rncO, wholly within the rnc coding region. Genetic analysis revealed the function of these structural elements. The single-stranded leader is not required for stability or RNase III control, stem-loop II is required only for RNase III control, and both stem-loops I and III are required for stability. Stem-loop II effectively serves only as the site at which RNase III cleaves to remove stem-loop I and thereby initiates decay, after which RNase III plays no role. Mutations at the cleavage site underscore the importance of base pairing for efficient RNase III attack. When stem-loops I and II were replaced with an artificial hairpin structure, stability was restored only partially, but was restored almost fully when a single-stranded leader was also added.