| Abstract: |
We have used DNA sequence affinity chromatography previously to purify a murine erythroid cell nuclear factor termed α-CP1. This promoter selective transcription factor is a heterotypic CCAAT factor composed of at least seven polypeptides with M(r) values that range from 27,000 to 38,000. Peptide mapping experiments reported here show that these seven polypeptides fall into three distinct classes (α, β, and γ). In addition chemical cross-linking, sedimentation, and gel filtration studies suggest that α-CP1 is a heterotrimeric factor composed of one polypeptide from each class. A core component of the factor (αβ) is stable at moderately high ionic strengths, whereas the γ polypeptides are more weakly associated with the particle. The native factor binds tightly to the α-globin CCAAT box (K(d) = 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 α-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 (K(d) = 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 α-CP1 to activate α-globin gene transcription in vitro. These latter results show that the DNA binding domain of α-CP1 is readily distinguished from the domains required to meddiate activation of gene transcription. |
| Keywords: |
mutation; dna-binding proteins; nonhuman; protein conformation; animal cell; mouse; animal; mice; protein dna binding; cell line; transcription factor; animalia; molecular sequence data; cell culture; dna, neoplasm; murinae; base sequence; binding site; cell nucleus; molecular weight; centrifugation, density gradient; ccaat-enhancer-binding proteins; chromatography, gel; peptide mapping; deoxyribonuclease i; oligonucleotide probes; priority journal; article; support, non-u.s. gov't; support, u.s. gov't, p.h.s.; erythroleukemia cell
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