EXPERIMENTAL FIGURE 9-24 DNase I footprinting reveals the region of a DNA sequence where a transcription factor binds. (a) A DNA fragment known to contain a transcription-control element is labeled at one end with ³²P (red dot). Portions of the labeled DNA sample are then digested with DNase I in the presence and in the absence of protein samples containing a sequence-specific DNA-binding protein. DNase I hydrolyzes the phosphodiester bonds of DNA between the 3′ oxygen on the deoxyribose of one nucleotide and the 5′ phosphate of the next nucleotide. A low concentration of DNase I is used so that, on average, each DNA molecule is cleaved just once (vertical arrows). If the protein sample does not contain a protein that binds to a specific sequence in the labeled DNA, the DNA fragment is cleaved at multiple positions between the labeled and unlabeled ends of the original fragment, as in sample A (left). If the protein sample does contain such a protein, as in sample B (right), the protein binds to its cognate sequence in the DNA, thereby protecting a portion of the fragment from digestion. Following DNase treatment, the DNA is separated from protein, denatured to separate the strands, and electrophoresed. Autoradiography of the resulting gel detects only labeled strands and reveals fragments extending from the labeled end to the site of cleavage by DNase I. Cleavage fragments containing the transcription-control element show up on the gel for sample A but are missing in sample B because the bound cognate protein has blocked cleavages within that sequence and thus production of the corresponding fragments. The missing bands on the gel constitute the footprint. (b) Footprints produced by increasing amounts of TBP (indicated by the triangle) and of TFIID on the strong adenovirus major late promoter.

[Part (b) from Zhou, Q. et al., “Holo-TFIID supports transcriptional stimulation by diverse activators and from a TATA-less promoter,” Genes & Development, 11/1992; 6(10):1964–74; republished with permission from Cold Spring Harbor Laboratory Press.]