HMG-I proteins are DNA-binding proteins thought to affect the formation and function of transcription complexes. Each protein contains three DNA-binding motifs, known as AT-hooks, that bind in the minor groove ofAT tracts in DNA. Multiple AT-hooks within a polypeptide chain should contact multiple AT tracts, but the rules governing these interactions have not been defined. In this study, we demonstrate that high-affinity binding uses two or three appropriately spaced AT tracts as a single multivalent binding site. These principles have implications for binding to regulatory elements such as the interferon ,8 enhancer, TATA boxes, and serum response elements.The HMG-I family of high mobility group chromosomal proteins consists of three members: HMG-I and HMG-Y, which are alternatively spliced products of a single gene (1-3), and HMGI-C, which is the product of a separate gene (4, 5). There is evidence that HMG-I(Y) is involved in the transcriptional regulation of genes, the best characterized example being the interferon ,B gene (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Specific biochemical functions of HMGI-C have not been elucidated, but recent reports of an HMGI-C knockout in the mouse causing the pygmy phenotype (18) and the implication of HMGI-C gene rearrangements in lipomas and other benign mesenchymal tumors (19) suggest an important role for this protein in cell growth and differentiation. Consistent with such a role is the activation of HMG-I genes as part of the delayed early response to growth factors (20).The HMG-I proteins are DNA-binding proteins, and the DNA-binding motif is known as an , which has been shown to bind in the minor groove of AT tracts of double-stranded DNA (22,23). Footprinting studies have shown that many AT tracts of 4, 5, 6, or more bp can be bound by HMG-I proteins (22). However, of the multitude of AT tracts in cellular DNA, such as TATA boxes, homeodomainbinding sites, serum response elements, matrix attachment regions, and many others, it is not clear which among them are high-affinity HMG-I protein-binding sites of functional significance.Since each HMG-I protein molecule contains three AThooks, each one is presumably able to mediate contact with an AT tract in DNA. This suggests the likelihood that highaffinity binding would involve multivalent binding of a single polypeptide to multiple AT tracts in close proximity to each other, although this form of binding has not been well defined. The purpose of the studies described in this report was to define multivalent, high-affinity binding sites for the HMG-I proteins.sequencing. HMGI-C was then subcloned into pET-3d (Novagen) and HMG-I and -Y were subcloned into pET-28d (Novagen), using Escherichia coli strain DHSa (GIBCO/BRL) to produce the plasmids pET/HMG-C, pET/HMG-I, and pET/HMG-Y, respectively.Protein Purification. The above plasmids were transferred into E. coli strain BL-21(DE3) (Novagen) and protein expression was induced with isopropyl thiogalactoside at 1 mM for 3 hr. Cells were harvested by centrifuga...