Magnesium-Agarose Electrophoretic Mobility Shift Assay (EMSA) of Transcription Factor IID Binding to DNA

Abstract

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.