He Chenlu, Custer Gregory, Wang Jingheng, Matysiak Silvina, Beckett Dorothy
Department of Chemistry & Biochemistry and ‡Fischell Department of Bioengineering, University of Maryland , College Park, Maryland 20742, United States.
Biochemistry. 2018 Feb 20;57(7):1119-1129. doi: 10.1021/acs.biochem.7b01122. Epub 2018 Feb 5.
Small molecules regulate transcription in both eukaryotes and prokaryotes by either enhancing or repressing assembly of transcription regulatory complexes. For allosteric transcription repressors, superrepressor mutants can exhibit increased sensitivity to small molecule corepressors. However, because many transcription regulatory complexes assemble in multiple steps, the superrepressor phenotype can reflect changes in any or all of the individual assembly steps. Escherichia coli biotin operon repression complex assembly, which responds to input biotin concentration, occurs via three coupled equilibria, including corepressor binding, holorepressor dimerization, and binding of the dimer to DNA. A genetic screen has yielded superrepressor mutants that repress biotin operon transcription in vivo at biotin concentrations much lower than those required by the wild type repressor. In this work, isothermal titration calorimetry and sedimentation measurements were used to determine the superrepressor biotin binding and homodimerization properties. The results indicate that, although all variants exhibit biotin binding affinities similar to that measured for BirA, five of the six superrepressors show altered homodimerization energetics. Molecular dynamics simulations suggest that the altered dimerization results from perturbation of an electrostatic network that contributes to allosteric activation of BirA for dimerization. Modeling of the multistep repression complex assembly for these proteins reveals that the altered sensitivity of the transcription response to biotin concentration is readily explained solely by the altered superrepressor homodimerization energetics. These results highlight how coupled equilibria enable alterations in a transcription regulatory response to input signal through an indirect mechanism.
小分子通过增强或抑制转录调节复合物的组装来调控真核生物和原核生物中的转录。对于变构转录抑制因子，超抑制突变体对小分子共抑制因子可能表现出更高的敏感性。然而，由于许多转录调节复合物是通过多个步骤组装而成的，超抑制表型可能反映了任何一个或所有单个组装步骤的变化。大肠杆菌生物素操纵子抑制复合物的组装对输入的生物素浓度有响应，它通过三个耦合平衡发生，包括共抑制因子结合、全抑制因子二聚化以及二聚体与DNA的结合。一项遗传筛选产生了超抑制突变体，这些突变体在体内能在比野生型抑制因子所需浓度低得多的生物素浓度下抑制生物素操纵子的转录。在这项研究中，使用等温滴定量热法和沉降测量来确定超抑制因子的生物素结合和同二聚化特性。结果表明，尽管所有变体都表现出与BirA测量值相似的生物素结合亲和力，但六个超抑制因子中的五个显示出同二聚化能量的改变。分子动力学模拟表明，二聚化的改变是由于一个静电网络的扰动所致，该网络有助于BirA二聚化的变构激活。对这些蛋白质的多步骤抑制复合物组装进行建模表明，转录反应对生物素浓度的敏感性改变仅通过超抑制因子同二聚化能量的改变就能很容易地解释。这些结果突出了耦合平衡如何通过间接机制实现转录调节反应对输入信号的改变。
