A quantitative analysis of nuclear factor I/DNA interactions

Abstract

Nuclear factor 1 (NFI) was purified to homogeneity from porcine liver by DNA-affinity chromatography and displays a single band with a molecular weight of 36 kDa in SDS-polyacrylamide gels. The purified protein was used to determine absolute equilibrium binding constants by gel retardation techiques for a variety of DNA fragments with genuine or mutated NFI binding sites and a number of DNA fragments derived from various eukaryotic promoters carrying the CCAAT-box, as a half-site for NFI binding. We present a model which allows prediction of the functional significance of mutated NFI binding-sites from sequence data. The data suggest that the single molecular species of NFI from porcine liver may not be able to recogize and activate the -CCAAT-promoter element in vivo without additional interactions, e.g. with other proteins.