Zerby D, Lieberman P M
Wistar Institute, Philadelphia, Pennsylvania 19104, USA.
Methods. 1997 Jul;12(3):217-23. doi: 10.1006/meth.1997.0474.
The general transcription factors TFIID and TFIIA are critical for the recognition of promoter start sites and mediate the stimulatory effect of some transcriptional activators. The regulation of TFIID binding to promoter DNA by activators and coactivators can be studied using a modified gel electrophoresis mobility shift assay (EMSA). TFIID is a multiprotein complex that consists of the TATA binding protein (TBP) and TBP associated factors (TAFs). TBP is a sequence-specific DNA binding protein that binds in the minor groove and introduces an energetically unfavorable bending angle of 100 degrees in the DNA. The activated preinitiation complex consists of TAFs, TBP, TFIIA, multiple activators, and approximately 200 bp of promoter DNA. The large mass and DNA distortions of the preinitiation complex preclude the use of conventional low ionic strength polyacrylamide gel EMSA for analysis. These large complexes can be analyzed by EMSA in agarose gels that contain magnesium ion. The Mg-agarose EMSA is a simple system useful for resolution of large multiprotein complexes that may introduce distortions in linear DNA. Important parameters are discussed so that this technique can be generally applied to other model activators.
通用转录因子TFIID和TFIIA对于启动子起始位点的识别至关重要，并介导一些转录激活因子的刺激作用。可使用改良的凝胶电泳迁移率变动分析（EMSA）研究激活因子和共激活因子对TFIID与启动子DNA结合的调控。TFIID是一种多蛋白复合物，由TATA结合蛋白（TBP）和TBP相关因子（TAFs）组成。TBP是一种序列特异性DNA结合蛋白，它结合在小沟中，并在DNA中引入一个能量上不利的100度弯曲角。活化的起始前复合物由TAFs、TBP、TFIIA、多种激活因子和大约200 bp的启动子DNA组成。起始前复合物的大质量和DNA扭曲使得无法使用传统的低离子强度聚丙烯酰胺凝胶EMSA进行分析。这些大复合物可通过含有镁离子的琼脂糖凝胶中的EMSA进行分析。Mg-琼脂糖EMSA是一个简单的系统，可用于解析可能在线性DNA中引入扭曲的大型多蛋白复合物。文中讨论了重要参数，以便该技术能够普遍应用于其他模型激活因子。
