Investigation of Changes in Tetracycline Repressor Binding upon Mutations in the Tetracycline Operator.
Investigation of Changes in Tetracycline Repressor Binding upon Mutations in the Tetracycline Operator.
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四环素操纵子突变后四环素阻遏物结合变化的研究。
DOI：
10.1021/je500225x
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发表时间：
2014
影响因子：
--
通讯作者：
Kaznessis,YiannisN
中科院分区：
文献类型：
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作者：
Bolintineanu,DanS;Volzing,Katherine;Vivcharuk,Victor;Sayyed-Ahmad,Abdallah;Srivastava,Poonam;Kaznessis,YiannisN
四环素操纵子是一种重要的基因网络元件，因其具有开关性质而被广泛应用于合成生物学中。该系统的核心是tet抑制蛋白(TetR)与其同源DNA序列(Teto)的高度特异性相互作用。TetR与Teto的结合实际上阻止了Teto下游基因的表达，因为它排除了RNA聚合酶与启动子的结合和启动转录。突变Teto序列改变了TetR-Teto结合的强度，从而为合成生物学家提供了操纵基因表达水平的工具。我们使用分子动力学(MD)模拟结合自由能微扰方法研究了TetR与不同Teto突变体的结合亲和力。我们还在大肠杆菌中进行了基于这些突变体的一系列启动子的体内测试。我们获得了实验绿色荧光蛋白(GFP)抑制水平与分子模拟计算的结合自由能差之间的合理一致。在所有情况下，野生型Teto序列产生最强的TetR结合，这在实验中，就GFP水平而言，在模拟中，在自由能变化方面都可以观察到。我们测试的四个Teto突变体中有两个产生了相对较强的结合，而另外两个突变体往往明显较弱。在我们自己的实验数据、之前用不同系统进行的实验以及从我们的模拟计算的自由能变化之间，这一Teto突变体子集的聚集和相对排名通常是一致的。总体而言，这项工作提供了对一个重要的合成生物系统的见解，并展示了分子模拟在定量解释生物相关行为方面的潜力和局限性。
The tetracycline operon is an important gene network component, commonly used in synthetic biology applications because of its switch-like character. At the heart of this system is the highly specific interaction of the tet repressor protein (TetR) with its cognate DNA sequence (tetO). TetR binding on tetO practically stops expression of genes downstream of tetO by excluding RNA polymerase from binding the promoter and initiating transcription. Mutating the tetO sequence alters the strength of TetR–tetO binding and thus provides a tool to synthetic biologists to manipulate gene expression levels. We employ molecular dynamics (MD) simulations coupled with the free energy perturbation method to investigate the binding affinity of TetR to different tetO mutants. We also carry out in vivo tests inEscherichia colifor a series of promoters based on these mutants. We obtain reasonable agreement between experimental green fluorescent protein (GFP) repression levels and binding free energy differences computed from molecular simulations. In all cases, the wild-type tetO sequence yields the strongest TetR binding, which is observed both experimentally, in terms of GFP levels, and in simulation, in terms of free energy changes. Two of the four tetO mutants we tested yield relatively strong binding, whereas the other two mutants tend to be significantly weaker. The clustering and relative ranking of this subset of tetO mutants is generally consistent between our own experimental data, previous experiments with different systems and the free energy changes computed from our simulations. Overall, this work offers insights into an important synthetic biological system and demonstrates the potential, as well as limitations of molecular simulations to quantitatively explain biologically relevant behavior.