Quantifying Translational Coupling in E. coli Synthetic Operons Using RBS Modulation and Fluorescent Reporters
Quantifying Translational Coupling in E. coli Synthetic Operons Using RBS Modulation and Fluorescent Reporters
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DOI：
10.1021/sb400002n
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发表时间：
2013-06-01
影响因子：
4.7
通讯作者：
Milo, Ron
中科院分区：
文献类型：
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作者：
Levin-Karp, Ayelet;Barenholz, Uri;Milo, Ron
翻译耦合是操纵子内编码的邻近基因翻译效率的相互依赖性。耦合程度可以通过测量基因的翻译速率如何被其上游基因的翻译速率调节来量化。几十年前在原核操纵子中观察到了平移耦合，但平移耦合导致的调制的定量范围以及控制这种调制的因素仅被部分表征。在这项研究中，我们使用携带荧光报告基因的质粒库系统地量化和表征大肠杆菌合成操纵子中的翻译耦合，这些荧光报告基因由一组不同的核糖体结合位点（RBS）序列控制。发现下游基因表达水平通过翻译偶联被上游基因表达增强，增强水平从几乎没有偶联到超过10倍，具体取决于上游基因的序列。此外，我们发现我们系统中操纵子第二个和第三个位置之间的平移耦合水平相似。耦合取决于上游基因的终止密码子和下游基因的起始密码子之间的距离。这项研究首次系统地、定量地描述了合成大肠杆菌操纵子中的翻译耦合。我们的分析将有助于合成生物学中基因表达的精确操作，并作为理解翻译表达调节机制的一步。
Translational coupling is the interdependence of translation efficiency of neighboring genes encoded within an operon. The degree of coupling may be quantified by measuring how the translation rate of a gene is modulated by the translation rate of its upstream gene. Translational coupling was observed in prokaryotic operons several decades ago, but the quantitative range of modulation translational coupling leads to and the factors governing this modulation were only partially characterized. In this study, we systematically quantify and characterize translational coupling in E. coli synthetic operons using a library of plasmids carrying fluorescent reporter genes that are controlled by a set of different ribosome binding site (RBS) sequences. The downstream gene expression level is found to be enhanced by the upstream gene expression via translational coupling with the enhancement level varying from almost no coupling to over 10-fold depending on the upstream gene's sequence. Additionally, we find that the level of translational coupling in our system is similar between the second and third locations in the operon. The coupling depends on the distance between the stop codon of the upstream gene and the start codon of the downstream gene. This study is the first to systematically and quantitatively characterize translational coupling in a synthetic E. coli operon. Our analysis will be useful in accurate manipulation of gene expression in synthetic biology and serves as a step toward understanding the mechanisms involved in translational expression modulation.