Footprinting

• hormone-receptor complexes that bind to their hormone response elements
• transcription factors that bind eukaryotic operators, enhancers, and silencers
• the lac repressor that shuts down the lac operon in E. coli [Discussion of the lac operon]

How, for example, does one determine the DNA sequence to which the lac repressor binds?

The procedure:

• Clone a piece of DNA that contains the operator site to which the repressor binds.
• Label one end of the DNA molecules with a radioactive molecule, e.g. radioactive ATP.
• Digest the DNA with DNase I.
  • DNase I cuts DNA molecules randomly (in contrast to restriction enzymes that cut where they find a particular sequence)
  • Choose such gentle conditions that most molecules will be cut only once.
• The result will be a mixture of radioactive fragments of varying length, with the smallest increment in length represented by a single nucleotide.
• Separate the fragments by electrophoresis.
• Binding of the lac repressor to the sequence of 24 base pairs in the operator prevents DNase I from attacking that region of the molecule.
• When the fragments are separated by electrophoresis, those representing the lengths covered by the repressor will be missing from the autoradiogram.
• The resulting gap is the "footprint".
• The same sample of DNA (unprotected by the repressor) is subjected to normal DNA sequencing and the resulting ladder aligned with the footprint autoradiogram.
• The exact sequence of bases in the lac operator can then be read directly because they represent the rungs of the ladder missing in the footprint.