Activation function 1 of glucocorticoid receptor binds TATA-binding protein in vitro and in vivo
Activation function 1 of glucocorticoid receptor binds TATA-binding protein in vitro and in vivo
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DOI：
10.1210/me.2005-0257
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发表时间：
2006-06-01
影响因子：
--
通讯作者：
Thompson, E. Brad
中科院分区：
文献类型：
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作者：
Copik, Alicja J.;Webb, M. Scott;Thompson, E. Brad
糖皮质激素受体（GR）刺激转录的机制尚不清楚，尽管很明显GR影响转录机制的组装。在这个过程中，tata结合蛋白（TBP）与TATA-box的结合被认为是必不可少的。众所周知，GR可以在体外与TBP相互作用，但TBP与GR的直接相互作用以前没有被定量表征，也没有被认为是组装转录复合物的重要步骤。在此，我们证明TBP-GR相互作用在功能上是显著的，通过组合技术表征了TBP和GR在体外的关联，并确认了这种相互作用在体内的作用。结合凝胶电泳、沉积平衡和等温微量热滴定，分析了TBP-GR相互作用的化学计量学、亲和性和热力学特征。TBP结合重组GR激活函数1 (AF1)，其化学计量比为1:2，解离常数在纳摩尔范围内。在体内荧光共振能量转移实验中，使用荧光标记的TBP和各种GR构建物，瞬时转染到CV-1细胞中，显示GR-TBP相互作用，依赖于AF1。AF1缺失变异体在共表达的青色荧光蛋白和黄色荧光蛋白水平上表现出荧光共振能量转移效率，表明相互作用依赖于AF1结构域。为了证明体内GR- TBP相互作用的功能作用，体内TBP表达量的增加刺激了GR驱动的报告基因和内源性基因的表达，并且这种作用也特异性依赖于AF1。
The mechanism through which the glucocorticoid receptor (GR) stimulates transcription is still unclear, although it is clear that the GR affects assembly of the transcriptional machinery. The binding of the TATA-binding protein (TBP) to the TATA-box is accepted as essential in this process. It is known that the GR can interact in vitro with TBP, but the direct interaction of TBP with GR has not been previously characterized quantitatively and has not been appreciated as an important step in assembling the transcriptional complex. Herein, we demonstrate that the TBP-GR interaction is functionally significant by characterizing the association of TBP and GR in vitro by a combination of techniques and confirming the role of this interaction in vivo. Combined analysis, using native gel electrophoresis, sedimentation equilibrium, and isothermal microcalorimetry titrations, characterize the stoichiometry, affinity, and thermodynamics of the TBP-GR interaction. TBP binds recombinant GR activation function 1 (AF1) with a 1:2 stoichiometry and a dissociation constant in the nanomolar range. In vivo fluorescence resonance energy transfer experiments, using fluorescently labeled TBP and various GR constructs, transiently transfected into CV-1 cells, show GR-TBP interactions, dependent on AF1. AF1-deletion variants showed fluorescence resonance energy transfer efficiencies on the level of coexpressed cyan fluorescent protein and yellow fluorescent protein, indicating that the interaction is dependent on AF1 domain. To demonstrate the functional role of the in vivo GR- TBP interaction, increased amounts of TBP expressed in vivo stimulated expression of GR- driven reporters and endogenous genes, and the effect was also specifically dependent on AF1.