Evidence of a protein-binding region in DNA

Protein-binding regions in DNA represent regulatory
areas; thus, their analysis can yield some
insights into gene regulation. Protein-binding
DNA regions can be demonstrated in several
ways. With band-shift analysis (1), proteinbound
and non-protein-bound DNA fragments
are differentiated using gel electrophoresis in
direction towards the small fragments, a DNA
fragment that is part of a DNA-protein complex
migrates more slowly than a free DNA fragment
of the same size. The DNA-protein complex is
found at a different position (“band shift”). DNA
footprinting (2) is another procedure for identifying
protein-binding sites on DNA. The principle
of DNA footprinting is that a proteinbound
DNA region, e.g., the polymerase-promoter
complex, is protected from the effects of
a DNA-cleaving enzyme (DNAase I). Previously
isolated DNA is cut into different fragments by
DNAase, and the fragments are sorted according
to size by gel electrophoresis. Since the DNA
protein-binding region is protected from cleavage
by DNAase I (DNAase I protection experiment),
DNA bands from the binding region are
missing (“footprint”).