The Role of RNA Structure in Determining RNase E-dependent Cleavage Sites in the MRNA for Ribosomal Protein S20 in Vitro

An RNA encompassing the 3' 147 residues of the mRNA for ribosomal protein S20 in Escherichia coli constitutes a naturally occurring degradative intermediate whose formation depends on RNase E. We have investigated the role of internal stem-loop structures in the RNase E-dependent cleavage which generates this product from S20 mRNA in a partially fractionated processing system in vitro. Individual stem-loops have been removed by deletion or destabilized by point mutations. No single hairpin structure is absolutely required for RNase E-dependent cleavage at the site 147 residues from the 3' end of the RNA. Primary sequences or secondary structures 5' or 3' to this site exert only a modest influence on the specificity of cleavage but can strongly modify its rate. Moreover, mutations in the S20 mRNA which destabilize stems 5' or 3' to the prominent cleavage site also reveal several strong cryptic RNase E cleavage sites. These data greatly strengthen the hypothesis that RNase E is a single-strand specific endoribonuclease. Our data further demonstrate that stem-loop structures adjacent to the prominent cleavage site are unlikely to provide a site of recognition for RNase E. Rather, they appear to stabilize (or "anchor") the local secondary structure so that the cleavage site is single-stranded and to occlude alternative sites so that the initial products of cleavage resist further attack.