title: Proteins binding to site C2 (muE3) in the immunoglobulin heavy-chain enhancer exist in multiple oligomeric forms.
abstract: We describe the purification to near homogeneity of proteins binding to site C2 (muE3) in the immunoglobulin heavy-chain enhancer. Proteins binding to this site produce four protein-DNA complexes which are distinguished by their mobility in gel retardation assays and their elution properties in an anion exchange column. DNA affinity-purified preparations of three chromatographically separated pools, containing different subsets of the four complexes, each contained three polypeptides of 42.5, 44, and 45 kilodaltons (kDa). UV crosslinking of protein to enhancer DNA demonstrated that site C2-binding activities in the three different pools bound DNA through proteins of similar sizes (about 45 kDa), even though the protein-DNA complexes formed by these binding activities were quite distinct. Gel exclusion chromatography and equilibrium binding analyses indicated that the distinct protein-DNA complexes were due to different oligomeric forms of the individual subunits and that a larger multimeric form bound with high affinity to the heavy-chain enhancer site C2, while a smaller species had a much lower affinity for heavy-chain enhancer sequences. Purified protein has been used to map high-affinity binding sites for site C2-binding proteins within an immunoglobulin heavy-chain promoter and at site KE3 in the kappa light-chain enhancer.
authors.affiliation: Molecular Biology Institute, School of Medicine, University of California, Los Angeles 90024.
authors.forename: C L
authors.initials: CL
authors.lastname: Peterson
authors.forename: K
authors.initials: K
authors.lastname: Calame
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005803:Genes, Immunoglobulin; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D011401:Promoter Regions, Genetic; D011487:Protein Conformation
chemical_list: D004268:DNA-Binding Proteins; D004247:DNA