Paired termini stabilize antisense RNAs and enhance conditional gene silencing in Escherichia coli.
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.
Nakashima
Nobutaka
N
Department of Cell and Molecular Biology, Programme for Genomics and Bioinformatics, Karolinska Institute Berzelius väg 35, 171 77 Stockholm, Sweden.
Tamura
Tomohiro
T
Good
Liam
L
D000084:Acetate Kinase / Q000235:genetics / Q000378:metabolism; D000085:Acetates / Q000378:metabolism; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics; D054889:Fatty Acid Synthase, Type II; D020868:Gene Silencing; D009876:Operon; D020871:RNA Stability; D016372:RNA, Antisense / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014260:Triclosan / Q000494:pharmacology; D001616:beta-Galactosidase / Q000235:genetics
D000085:Acetates; D029968:Escherichia coli Proteins; D016372:RNA, Antisense; D011994:Recombinant Proteins; D014260:Triclosan; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); C493924:fabI protein, E coli; D000084:Acetate Kinase; D001616:beta-Galactosidase; D054889:Fatty Acid Synthase, Type II