Nucleic Acids Research Advance Access originally published online on October 24, 2006
Nucleic Acids Research 2006 34(20):e138; doi:10.1093/nar/gkl697
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Nucleic Acids Research, 2006, Vol. 34, No. 20 e138
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Paired termini stabilize antisense RNAs and enhance conditional gene silencing in Escherichia coli
1 Department of Cell and Molecular Biology, Programme for Genomics and Bioinformatics, Karolinska Institute Berzelius väg 35, 171 77 Stockholm, Sweden 2 Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST) 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, 062-8517 Sapporo, Japan 3 Laboratory of Molecular Environmental Microbiology, Graduate School of Agriculture, Hokkaido University Kita-9, Nishi-9, Kita-ku, 060-8589 Sapporo, Japan
*To whom correspondence should be addressed. Tel: +46 8 5248 6385, Fax: +46 8 32 39 50; Email: liam.good{at}ki.se
Received July 11, 2006. Revised August 21, 2006. Accepted September 8, 2006.
Reliable methods for conditional gene silencing in bacteria have been elusive. To improve silencing by expressed antisense RNAs (asRNAs), we systematically altered several design parameters and targeted multiple reporter and essential genes in Escherichia coli. A paired termini (PT) design, where flanking inverted repeats create paired dsRNA termini, proved effective. PTasRNAs targeted against the ackA gene within the acetate kinase-phosphotransacetylase operon (ackA-pta) triggered target mRNA decay and a 78% reduction in AckA activity with high genetic penetrance. PTasRNAs are abundant and stable and function through an RNase III independent mechanism that requires a large stoichiometric excess of asRNA. Conditional ackA silencing reduced carbon flux to acetate and increased heterologous gene expression. The PT design also improved silencing of the essential fabI gene. Full anti-fabI PTasRNA induction prevented growth and partial induction sensitized cells to a FabI inhibitor. PTasRNAs have potential for functional genomics, antimicrobial discovery and metabolic flux control.
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