Ectopic RNase E sites promote bypass of 5 '-end-dependent mRNA decay in Escherichia coli

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 me leader or 9S pre-rRNA into the 5'-non-coding region of the rpsTmRNA 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.