Purification of transcription cofactor complex CRSP.
Transcription of protein coding genes in metazoans involves the concerted action of enhancer binding proteins and the RNA polymerase II apparatus. The cross talk between these two classes of transcription factors is mediated by an elaborate set of cofactor complexes. For the activation of transcription by the promoter specificity protein 1 (Sp1), TATA binding protein-associated factors in the TFIID complex originally were identified as necessary coactivators, but the identity of additional cofactors required for activated transcription was unknown. Recently, we have reported the isolation and properties of a cofactor complex, CRSP (cofactor required for Sp1), which functions in conjunction with the TATA binding protein-associated factors to promote efficient activation of transcription by Sp1. CRSP contains unique subunits as well as polypeptides that are shared with other cofactor complexes. Here, we report a detailed purification protocol for the isolation of CRSP from human HeLa cells. Our purification strategy takes advantage of the ability of CRSP to bind Ni2+-nitrilotriacetic acid-agarose resin as well as other conventional chromatographic resins. We also describe a streamlined purification protocol that allows a more rapid and efficient means to isolate active CRSP.
Ryu
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Howard Hughes Medical Institute, University of California, Molecular and Cell Biology, 401 Barker Hall, Berkeley, CA 94720-3204, USA.
Tjian
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D002846:Chromatography, Affinity / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D056892:Mediator Complex; D016329:Sp1 Transcription Factor / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
C117286:CRSP protein, human; C492157:CRSP5 protein, human; C492160:MED14 protein, human; C503845:MED24 protein, human; D046911:Macromolecular Substances; D056892:Mediator Complex; D016329:Sp1 Transcription Factor; D015534:Trans-Activators