Sharma Vandana, Yamamura Asami, Yokobayashi Yohei
Department of Biomedical Engineering, University of California, Davis, 451 Health Sciences Drive, Davis, CA 95616, USA.
ACS Synth Biol. 2012 Jan 20;1(1):6-13. doi: 10.1021/sb200001q. Epub 2011 Aug 29.
It has become increasingly evident that noncoding small RNAs (sRNAs) play a significant and global role in bacterial gene regulation. A majority of the trans-acting sRNAs in bacteria interact with the 5' untranslated region (UTR) and/or the translation initiation region of the targeted mRNAs via imperfect base pairing, resulting in reduced translation efficiency and/or mRNA stability. Additionally, bacterial sRNAs often contain distinct scaffolds that recruit RNA chaperones such as Hfq to facilitate gene regulation. In this study, we describe a strategy to engineer artificial sRNAs that can regulate desired endogenous genes in Escherichia coli. Using a fluorescent reporter gene that was translationally fused to a native 5' mRNA leader sequence, active artificial sRNAs were screened from libraries in which natural sRNA scaffolds were fused to a randomized antisense domain. Artificial sRNAs that posttranscriptionally repress two endogenous genes ompF and fliC were isolated and characterized. We anticipate that the artificial sRNAs will be useful for dynamic control and fine-tuning of endogenous gene expression in bacteria for applications in synthetic biology.
越来越明显的是，非编码小RNA（sRNA）在细菌基因调控中发挥着重要且全面的作用。细菌中大多数反式作用sRNA通过不完全碱基配对与靶标mRNA的5'非翻译区（UTR）和/或翻译起始区相互作用，导致翻译效率降低和/或mRNA稳定性降低。此外，细菌sRNA通常包含独特的支架结构，可招募诸如Hfq之类的RNA伴侣以促进基因调控。在本研究中，我们描述了一种设计人工sRNA的策略，该策略可调控大肠杆菌中所需的内源基因。使用与天然5'mRNA前导序列翻译融合的荧光报告基因，从天然sRNA支架与随机反义结构域融合的文库中筛选出活性人工sRNA。分离并鉴定了在转录后抑制两个内源基因ompF和fliC的人工sRNA。我们预计，人工sRNA将有助于对细菌中的内源基因表达进行动态控制和微调，以用于合成生物学应用。
