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

Authors

  • Kristian E. Baker,

    1. Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada V6T 1Z3.
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    • Present address: Howard Hughes Medical Institute and Department of Molecular and Cellular Biology, University of Arizona, Room 403 Life Sciences South, PO Box 210106, Tucson, AZ 85721–0106, USA.

  • George A. Mackie

    Corresponding author
    1. Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada V6T 1Z3.
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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.

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