PROTEIN FOOTPRINTING REVEALS SPECIFIC BINDING MODES OF A HIGH-MOBILITY GROUP PROTEIN-I TO DNAS OF DIFFERENT CONFORMATION

The high mobility group proteins I and Y (HMGI/Y) are abundant compone nts of chromatin. They are thought to derepress chromatin, affect the assembly and activity of the transcriptional machinery, and associate with constitutive heterochromatin during mitosis. HMGI/Y protein molec ules contain three potential DNA-binding. motifs (AT-hooks), but the e xtent of contacts between DNA and the entire protein has not been dete rmined, We have used a protein-footprinting procedure to map regions o f the Chironomus HMGI protein molecule that are involved in contacts w ith DNA, We find that in the presence of double-stranded DNA all AT-ho ok motifs are protected against hydroxyl radical proteolysis. In contr ast, only two motifs were protected in the presence of four-wag juncti on DNA. Large regions that flank the AT-hook motifs were found to be s trongly protected against proteolysis in complexes with interferon-bet a promoter DNA, suggesting amino acid residues outside the AT-hooks co nsiderably contribute to DNA binding.