Improvement of the reverse tetracycline transactivator by single amino acid substitutions that reduce leaky target gene expression to undetectable levels.
Improvement of the reverse tetracycline transactivator by single amino acid substitutions that reduce leaky target gene expression to undetectable levels.
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DOI：
10.1038/srep27697
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发表时间：
2016-06-21
影响因子：
4.6
通讯作者：
Kærn M
中科院分区：
文献类型：
--
作者：
Roney IJ;Rudner AD;Couture JF;Kærn M
能够实现可诱导和可逆转录控制的条件基因表达系统是重要的研究工具，在生物医学和生物技术中具有广泛的应用。反向四环素转录激活剂是用于工程化基因表达控制的规范系统，能够对从酵母到人类细胞系和转基因动物的真核生物中的靶基因转录进行分级和无偿调节。然而，即使在没有四环素的情况下，该系统也有激活转录的倾向，并且这种泄漏的靶基因表达阻碍了其使用。在这里，我们确定了单个氨基酸取代，通过将渗漏转录减少到不可检测的水平，同时在诱导物存在的情况下保持高表达能力，大大增强了酵母系统的动态范围。虽然突变增加了完全诱导所需的诱导剂浓度，但额外的敏感性增强突变可以补偿这种效应，并赋予系统高度的鲁棒性。新型反式激活蛋白变体将在基因表达的严格和可调调节至关重要的应用中有用。
Conditional gene expression systems that enable inducible and reversible transcriptional control are essential research tools and have broad applications in biomedicine and biotechnology. The reverse tetracycline transcriptional activator is a canonical system for engineered gene expression control that enables graded and gratuitous modulation of target gene transcription in eukaryotes from yeast to human cell lines and transgenic animals. However, the system has a tendency to activate transcription even in the absence of tetracycline and this leaky target gene expression impedes its use. Here, we identify single amino-acid substitutions that greatly enhance the dynamic range of the system in yeast by reducing leaky transcription to undetectable levels while retaining high expression capacity in the presence of inducer. While the mutations increase the inducer concentration required for full induction, additional sensitivity-enhancing mutations can compensate for this effect and confer a high degree of robustness to the system. The novel transactivator variants will be useful in applications where tight and tunable regulation of gene expression is paramount.