Carey Michael F, Peterson Craig L, Smale Stephen T
Cold Spring Harb Protoc. 2010 Nov 1;2010(11):pdb.prot5514. doi: 10.1101/pdb.prot5514.
The general transcription factor IID (TFIID) is a key target for regulation because its binding to a core promoter is the nucleating step in transcription complex assembly. Many eukaryotic activators stimulate recruitment of the TFIID when its concentration is made limiting at a promoter in vitro. Magnesium-agarose gels can separate large complexes containing TFIID, TFIIA (the DA complex), and TFIIB (the DAB complex) and permit a quantitative measurement of how activators stimulate assembly of such complexes. The advantage of the electrophoretic mobility shift assay (EMSA) is that the reactions can be performed under subsaturating conditions where a TFIID footprint might not be observed. Typically, the activator is incubated with a 32P-labeled DNA template, recombinant TFIIA purified from Escherichia coli, and immunopurified TFIID. After incubation, the samples are electrophoresed on magnesium-containing agarose gels, dried onto DEAE-cellulose paper, and autoradiographed. The DNA-protein complexes containing TFIID migrate with reduced mobility on magnesium-agarose gels both because of the large size of the complex and because the TATA-binding protein (TBP) subunit induces a sharp bend in the DNA, causing altered mobility. By comparing the binding of TFIID over a wide concentration range, with and without activator, one can assess whether the activator interacts with TBP or with one of the TBP-associated factors (TAFIIs). Additional factors such as TFIIA and TFIIB can be added subsequently to quantify their contributions to assembly of the transcription complex.
通用转录因子IID（TFIID）是调控的关键靶点，因为它与核心启动子的结合是转录复合物组装的起始步骤。在体外，当启动子处TFIID的浓度受到限制时，许多真核激活剂会刺激TFIID的募集。镁 - 琼脂糖凝胶可以分离包含TFIID、TFIIA（DA复合物）和TFIIB（DAB复合物）的大复合物，并能定量测量激活剂如何刺激此类复合物的组装。电泳迁移率变动分析（EMSA）的优点是反应可以在亚饱和条件下进行，此时可能观察不到TFIID足迹。通常，将激活剂与32P标记的DNA模板、从大肠杆菌中纯化的重组TFIIA以及免疫纯化的TFIID一起孵育。孵育后，将样品在含镁的琼脂糖凝胶上进行电泳，干燥到DEAE - 纤维素纸上，然后进行放射自显影。含有TFIID的DNA - 蛋白质复合物在镁 - 琼脂糖凝胶上迁移率降低，这既是因为复合物尺寸大，也是因为TATA结合蛋白（TBP）亚基会使DNA产生急剧弯曲，导致迁移率改变。通过比较在有无激活剂的情况下，TFIID在宽浓度范围内的结合情况，可以评估激活剂是与TBP相互作用，还是与TBP相关因子（TAFIIs）之一相互作用。随后可以添加其他因子，如TFIIA和TFIIB，以量化它们对转录复合物组装的贡献。
