Purification and analysis of RNA polymerase II transcription factors by using wheat germ agglutinin affinity chromatography.
We recently found that many RNA polymerase II transcription factors are modified with N-acetylglucosamine residues. These sugar moieties confer upon transcription factors an ability to bind the lectin wheat germ agglutinin. We have taken advantage of this interaction to devise a purification procedure for the "GC-box" binding transcription factor Sp1. Crude nuclear extracts are first subjected to wheat germ agglutinin affinity chromatography and then subjected to sequence-specific DNA affinity chromatography. The Sp1 protein purified by this procedure is at least 95% pure, and the overall recovery is greater than 80%. In addition to yielding larger quantities of Sp1 than conventional schemes, the new purification procedure is also simpler and more rapid. We show that wheat germ agglutinin affinity chromatography can also be used to purify the glycosylated forms of the CCAAT-binding transcription factor. Thus, wheat germ agglutinin affinity chromatography may aid the purification of other transcription factors that bear N-acetylglucosamine residues. Furthermore, the ability to separate glycosylated forms of transcription factors from their unglycosylated counterparts by wheat germ agglutinin affinity chromatography should facilitate investigations into the role of N-acetylglucosamine residues in the functioning of transcription factor proteins.
Jackson
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SP
Howard Hughes Medical Institute, Department of Biochemistry, University of California, Berkeley 94720.
Tjian
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D000117:Acetylglucosamine / Q000032:analysis; D022762:CCAAT-Enhancer-Binding Proteins; D002467:Cell Nucleus / Q000032:analysis; D002846:Chromatography, Affinity; D004247:DNA; D004268:DNA-Binding Proteins / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D012319:RNA Polymerase II / Q000378:metabolism*; D016329:Sp1 Transcription Factor; D014157:Transcription Factors / Q000302:isolation & purification* / Q000378:metabolism; D014909:Wheat Germ Agglutinins; D051840:Y-Box-Binding Protein 1
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D014909:Wheat Germ Agglutinins; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D004247:DNA; D012319:RNA Polymerase II; D000117:Acetylglucosamine