Exploring the sequence space for tetracycline-dependent transcriptional activators: Novel mutations yield expanded range and sensitivity
Exploring the sequence space for tetracycline-dependent transcriptional activators: Novel mutations yield expanded range and sensitivity
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DOI：
10.1073/pnas.130192197
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发表时间：
2000-07-05
影响因子：
11.1
通讯作者：
Hillen, W
中科院分区：
文献类型：
--
作者：
Urlinger, S;Baron, U;Hillen, W
控制大肠杆菌中四环素抗性的调节元件先前被转化为在多种真核细胞中发挥作用的高度特异性的转录调节系统。一个四环素阻遏物（TetR）突变体产生了rtTA，这是一种四环素控制的反式激活因子，需要多西环素（Dox）与泰特操纵子结合，从而激活P-tet启动子。尽管rtTA的有趣的特性，它的使用是有限的，特别是在转基因动物中，因为它在一些器官中相对低效的多西环素诱导，它的不稳定性，和它的残留亲和力tetO在没有Dox，导致升高的背景活动的目标启动子。为了消除这些限制，我们已经诱变tTA DNA，并在酿酒酵母中选择具有降低的基础活性和增加的Dox敏感性的rtTA突变体。鉴定了五种新的rtTA，其中两种具有大大改善的性质。最有前途的新反式激活因子rtTA 2（S）-M2在比rtTA低10倍的Dox浓度下起作用，在真核细胞中更稳定，并且在没有Dox的情况下不引起背景表达。优化与最小激活结构域融合的新反向TetR突变体的编码序列以在人细胞中表达并合成。所得到的反式激活因子允许在稳定转染的HeLa细胞中在4至5个数量级的范围内严格调节靶基因。这些rtTA版本将表达控制的紧密性与广泛的调控范围相结合，如先前广泛应用的tTA所示。
Regulatory elements that control tetracycline resistance in Escherichia coli were previously converted into highly specific transcription regulation systems that function in a wide variety of eukaryotic cells. One tetracycline repressor (TetR) mutant gave rise to rtTA, a tetracycline-controlled transactivator that requires doxycycline (Dox) for binding to tet operators and thus for the activation of P-tet promoters. Despite the intriguing properties of rtTA, its use was limited, particularly in transgenic animals, because of its relatively inefficient inducibility by doxycycline in some organs, its instability, and its residual affinity to tetO in absence of Dox, leading to elevated background activities of the target promoter. To remove these limitations, we have mutagenized tTA DNA and selected in Saccharomyces cerevisiae for rtTA mutants with reduced basal activity and increased Dox sensitivity. Five new rtTAs were identified, of which two have greatly improved properties. The most promising new transactivator, rtTA2(S)-M2, functions at a 10-fold lower Dox concentration than rtTA, is more stable in eukaryotic cells, and causes no background expression in the absence of Dox. The coding sequences of the new reverse TetR mutants fused to minimal activation domains were optimized for expression in human cells and synthesized. The resulting transactivators allow stringent regulation of target genes over a range of 4 to 5 orders of magnitude in stably transfected HeLa cells. These rtTA versions combine tightness of expression control with a broad regulatory range, as previously shown for the widely applied tTA.