RNase E cleaves at multiple sites in bubble regions of RNA I stem loops yielding products that dissociate differentially from the enzyme

Earlier work has shown that RNase E cleaves RNAI, the antisense repressor of replication of ColE1-type plasmids, producing pRNAI(-5), whose further decay is mediated by the poly(A)-dependent activity of polynucleotide phosphorylase and other 3' to 5' exonucleases. Using a poly(A) polymerase-deficient strain to impede exonucleolytic decay, we show that RNAI is additionally cleaved by RNase E at multiple sites, generating a series of decay intermediates that are differentially retained by the RNA binding domain (RBD) of RNase E. Primer extension analysis of RNAI decay intermediates and RNase T-1 mapping of the cleavage products of RNAI generated in vitro by affinity-purified RNase E showed that RNase E can cleave internucleotide bonds in the bubble regions of duplex RNA segments and in single-stranded regions. Chemical in situ probing of a complex formed between RNAI and the RBD indicates that binding to the RBD destabilizes RNAI secondary structure. Our results suggest a model in which a series of sequential RNase E-mediated cleavages occurring at multiple sites of RNAI, some of which may be made more accessible to RNase E by the destabilizing effects of its RBD, generate RNA fragments that are further degraded by poly(A)-dependent 3' to 5' exonucleases.