Expression and purification of the leucine zipper and DNA-binding domains of Fos and Jun: both Fos and Jun contact DNA directly.
The protein products of the fos and jun protooncogenes interact cooperatively in the form of a heterodimer with the activator protein 1 (AP-1) regulatory element. To characterize the properties of these proteins, we have expressed polypeptides comprised of the dimerization and DNA-binding domains of Fos and Jun in Escherichia coli. The mini-Fos (wbFos) and the mini-Jun (wbJun) proteins were purified to apparent homogeneity by using a nickel affinity chromatography procedure. Purified wbFos and wbJun associated rapidly in vitro and interacted cooperatively with the human metallothionein IIA AP-1-binding site. However, efficient DNA binding of wbJun and wbFos-wbJun complexes required an additional activity present in nuclear extracts. This activity was sensitive to alkylating agents and could be partially mimicked by the presence of reducing and stabilizing agents. DNase I footprinting experiments demonstrated that Jun homodimeric complexes and Fos-Jun heterodimeric complexes interacted with the same site on the human metallothionein IIA gene. Moreover, UV-crosslinking studies demonstrated that Fos and Jun contact DNA directly and that both proteins interacted equivalently with either strand of the AP-1-binding site.
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Department of Molecular Oncology and Virology, Roche Institute of Molecular Biology, Nutley, NJ 07110.
Luk
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Gentz
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Rauscher
F J
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Curran
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007930:Leucine; D008969:Molecular Sequence Data; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011519:Proto-Oncogenes; D051381:Rats; D015183:Restriction Mapping; D014157:Transcription Factors / Q000235:genetics*
D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D014157:Transcription Factors; D011505:Protein-Tyrosine Kinases; D007930:Leucine