Abstract

RNase E is an essential enzyme in Escherichia coli. The cleavage site of this single-stranded specific endoribonuclease is well-characterized in many RNA substrates. Here, we report that the upregulation of RNase E cleavage activity by a mutation that affects either RNA binding (Q36R) or enzyme multimerization (E429G) was accompanied by relaxed cleavage specificity. Both mutations led to enhanced RNase E cleavage in RNA I, an antisense RNA of ColE1-type plasmid replication, at a major site and other cryptic sites. Expression of a truncated RNA I with a major RNase E cleavage site deletion at the 5′-end (RNA I- 5) resulted in an approximately twofold increase in the steady-state levels of RNA I- 5 and the copy number of ColE1-type plasmid in E. coli cells expressing wild-type or variant RNase E compared to those expressing RNA I. These
results
indicate that RNA I- 5 does not efficiently function as an antisense RNA despite having a triphosphate group at the 5′-end, which protects the RNA from ribonuclease attack. Our study suggests that increased cleavage rates of RNase E lead to relaxed cleavage specificity on RNA I and the inability of the cleavage product of RNA I as an antisense regulator in vivo does not stem from its instability by having 5′-monophosphorylated end.

• Keywords: divergent promoter;enterohemorrhagic E. coli O157:H7;transcriptional activity;type VI secretion system