Levin-Karp Ayelet, Barenholz Uri, Bareia Tasneem, Dayagi Michal, Zelcbuch Lior, Antonovsky Niv, Noor Elad, Milo Ron
Department of Plant Sciences, Weizmann Institute of Science , Rehovot 76100, Israel.
ACS Synth Biol. 2013 Jun 21;2(6):327-36. doi: 10.1021/sb400002n. Epub 2013 Apr 16.
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.
翻译偶联是指操纵子内相邻基因的翻译效率之间的相互依赖性。偶联程度可以通过测量一个基因的翻译速率如何被其上游基因的翻译速率调节来进行量化。几十年前就在原核生物操纵子中观察到了翻译偶联现象，但翻译偶联导致的调节定量范围以及控制这种调节的因素仅得到了部分表征。在本研究中，我们使用携带由一组不同核糖体结合位点（RBS）序列控制的荧光报告基因的质粒文库，系统地量化和表征了大肠杆菌合成操纵子中的翻译偶联。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，增强水平根据上游基因的序列从几乎无偶联到超过10倍不等。此外，我们发现在我们的系统中，操纵子中第二个和第三个位置之间的翻译偶联水平相似。偶联取决于上游基因的终止密码子与下游基因的起始密码子之间的距离。本研究首次系统地、定量地表征了合成大肠杆菌操纵子中的翻译偶联。我们的分析将有助于在合成生物学中精确操纵基因表达，并朝着理解翻译表达调节所涉及的机制迈出了一步。
