Ectopic RNase E sites promote bypass of 5′‐end‐dependent mRNA decay in Escherichia coli

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Abstract
Summary: In Escherichia coli, 5′‐terminal stem–loops form major impediments to mRNA decay, yet conditions that determine their effectiveness or the use of alternative decay pathway(s) are unclear. A synthetic 5′‐terminal hairpin stabilizes the rpsT mRNA sixfold. This stabilization is dependent on efficient translational initiation and ribosome transit through at least two‐thirds of the coding sequence past a major RNase E cleavage site in the rpsT mRNA. Insertion of a 12–15 residue ‘ectopic’ RNase E cleavage site from either the rne leader or 9S pre‐rRNA into the 5′‐non‐coding region of the rpsT mRNA significantly reduces the stabilizing effect of the terminal stem–loop, dependent on RNase E. A similar insertion into the rpsT coding sequence is partially destabilizing. These findings demonstrate that RNase E can bypass an interaction with the 5′‐terminus, and exploit an alternative ‘internal entry’ pathway. We propose a model for degradation of the rpsT mRNA, which explains the hierarchy of protection afforded by different 5′‐termini, the use of internal entry for bypass of barriers to decay, ‘ectopic sites’ and the role of translating ribosomes.