Journal of Microbiology

Journal Article

Relaxed Cleavage Specificity of Hyperactive Variants of Escherichia coli RNase E on RNA I: Dayeong Bae , Hana Hyeon , Eunkyoung Shin , Ji&# , Kangseok Lee; J. Microbiol. 2023;61(2):211-220. Published online February 22, 2023; DOI: https://doi.org/10.1007/s12275-023-00013-z

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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.

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Engineering an Escherichia coli based in vivo mRNA manufacturing platform
Edward Curry, George Muir, Jixin Qu, Zoltán Kis, Martyn Hulley, Adam Brown
Biotechnology and Bioengineering.2024; 121(6): 1912. CrossRef

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