Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, Missouri 63110, USA.
Department of Biology, Washington University, St Louis, Missouri 63105, USA.
Nat Commun. 2017 Jan 20;8:14112. doi: 10.1038/ncomms14112.
Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels.
弱突变是基因功能遗传分析和合成生物学应用的一种有价值的工具。然而，目前产生弱突变的方法仅限于特定的生物体，不可预测地改变基因表达，或者依赖于目标基因时空表达的变化。在这里，我们提出了一种简单且可预测的方法，通过靶向翻译延伸来在模式生物中产生弱突变。在感兴趣的基因编码序列中添加连续的腺苷核苷酸，即所谓的 polyA 轨迹，会降低翻译延伸效率，在所有测试的细胞培养物和模式生物中，这会降低 mRNA 稳定性和蛋白质表达。我们表明，蛋白质表达是可调节的，与启动子强度无关，并可以通过改变 polyA 轨迹的序列特征进一步调节。这些特性使得该方法具有高度的可预测性和可操作性，可用于生成具有一系列表达水平的可编程等位基因系列。
