Magnesium-agarose electrophoretic mobility shift assay (EMSA) of transcription factor IID binding to DNA.
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.
Michael F
MF
Carey
Craig L
CL
Peterson
Stephen T
ST
Smale
D001345:Autoradiography / Q000379:methods; D001671:Biochemistry / Q000379:methods*; D004247:DNA / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005057:Eukaryotic Cells; D008274:Magnesium / Q000378:metabolism*; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D013194:Staining and Labeling / Q000379:methods; D035362:Transcription Factor TFIID / Q000378:metabolism*
D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D035362:Transcription Factor TFIID; D004247:DNA; D008274:Magnesium