RNASE-III AUTOREGULATION - STRUCTURE AND FUNCTION OF RNCO, THE POSTTRANSCRIPTIONAL OPERATOR

Expression of the Escherichia coli mc-era-recO operon is regulated pos ttranscriptionally by ribonuclease III (RNase Ill), encoded in the me gene. RNase III initiates rapid decay of the me operon mRNA by cleavin g a double-stranded region of the me leader. This region, termed rncO, is portable, conferring stability and RNase III regulation to heterol ogous RNAs. Here, we report the detailed analysis of mcO structure and function. The first 215 nt of the me leader are sufficient for its fu nction. Dimethylsulfate (DMS) modification in vivo revealed distinct s tructural elements in this region: a 13-nt single-stranded 5' leader, followed by a 6-bp stem-loop structure (I), a larger stem-loop structu re (II) containing the RNase III site, a single-stranded region contai ning the me translation initiation site, and a small stem-loop structu re (III) at the 3' terminus of mcO, wholly within the me 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 bot h stem-loops I and III are required for stability. Stem-loop It effect ively serves only as the site at which RNase III cleaves to remove ste m-loop I and thereby initiates decay, after which RNase III plays no r ole. 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 l eader was also added.