Paired termini stabilize antisense RNAs and enhance conditional gene silencing in Escherichia coli.
Paired termini stabilize antisense RNAs and enhance conditional gene silencing in Escherichia coli.
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DOI：
10.1093/nar/gkl697
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发表时间：
2006
影响因子：
14.9
通讯作者：
Good L
中科院分区：
文献类型：
--
作者：
Nakashima N;Tamura T;Good L
细菌中条件性基因沉默的可靠方法一直难以捉摸。为了改善表达的反义RNA(AsRNAs)的沉默作用，我们系统地改变了几个设计参数，并针对多个报告基因和必需基因在大肠杆菌中进行了定位。事实证明，成对末端(PT)设计是有效的，其中侧翼反向重复序列创建了成对的dsRNA末端。针对醋酸激酶-磷酸转乙酰酶操纵子(ackA-PTA)中ackA基因的PTasRNAs可引起靶mRNA的降解，并使AckA活性降低78%，具有较高的遗传外显性。PTasRNAs是丰富和稳定的，通过RNase III独立的机制发挥功能，该机制需要大量化学计量过剩的asRNA。条件性ackA沉默减少了向乙酸盐的碳流，增加了异源基因的表达。PT设计还改善了必需的Fabi基因的沉默。完全的抗Fabi PTasRNA诱导阻止生长，部分诱导使细胞对Fabi抑制剂敏感。PTasRNAs在功能基因组学、抗菌药物发现和代谢流量控制等方面具有潜在的应用前景。
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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