Physical characterization of the purified CCAAT transcription factor, alpha-CP1.
We have used DNA sequence affinity chromatography previously to purify a murine erythroid cell nuclear factor termed alpha-CP1. This promoter selective transcription factor is a heterotypic CCAAT factor composed of at least seven polypeptides with Mr values that range from 27,000 to 38,000. Peptide mapping experiments reported here show that these seven polypeptides fall into three distinct classes (alpha, beta, and gamma). In addition chemical cross-linking, sedimentation, and gel filtration studies suggest that alpha-CP1 is a heterotrimeric factor composed of one polypeptide from each class. A core component of the factor (alpha beta) is stable at moderately high ionic strengths, whereas the gamma polypeptides are more weakly associated with the particle. The native factor binds tightly to the alpha-globin CCAAT box (Kd = 5.71 x 10(-11 M), and mutational studies show that the DNA recognition site resides in a sequence decamer. DNA binding is significantly stabilized, however, by apparently nonspecific sequences 3' of the CCAAT recognition motif. Finally, the DNA binding domain of purified alpha-CP1 is moderately stable to protease digestion, a feature characteristic of heterotypic CCAAT factors. The proteolyzed factor has a slightly higher affinity for the CCAAT box (Kd = 2.8 x 10(-11) M), and its footprint cannot be distinguished from that of the intact factor. In contrast protease treatment abolishes the ability of alpha-CP1 to activate alpha-globin gene transcription in vitro. These latter results show that the DNA binding domain of alpha-CP1 is readily distinguished from the domains required to mediate activation of gene transcription.
Kim
C G
CG
DeWitt Wallace Research Laboratory, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Sheffery
M
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D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification; D003850:Deoxyribonuclease I; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D015345:Oligonucleotide Probes; D010449:Peptide Mapping; D011487:Protein Conformation
D022762:CCAAT-Enhancer-Binding Proteins; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D003850:Deoxyribonuclease I