9.7: Footprinting
- Page ID
- 4886
\( \newcommand{\vecs}[1]{\overset { \scriptstyle \rightharpoonup} {\mathbf{#1}} } \) \( \newcommand{\vecd}[1]{\overset{-\!-\!\rightharpoonup}{\vphantom{a}\smash {#1}}} \)\(\newcommand{\id}{\mathrm{id}}\) \( \newcommand{\Span}{\mathrm{span}}\) \( \newcommand{\kernel}{\mathrm{null}\,}\) \( \newcommand{\range}{\mathrm{range}\,}\) \( \newcommand{\RealPart}{\mathrm{Re}}\) \( \newcommand{\ImaginaryPart}{\mathrm{Im}}\) \( \newcommand{\Argument}{\mathrm{Arg}}\) \( \newcommand{\norm}[1]{\| #1 \|}\) \( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\) \( \newcommand{\Span}{\mathrm{span}}\) \(\newcommand{\id}{\mathrm{id}}\) \( \newcommand{\Span}{\mathrm{span}}\) \( \newcommand{\kernel}{\mathrm{null}\,}\) \( \newcommand{\range}{\mathrm{range}\,}\) \( \newcommand{\RealPart}{\mathrm{Re}}\) \( \newcommand{\ImaginaryPart}{\mathrm{Im}}\) \( \newcommand{\Argument}{\mathrm{Arg}}\) \( \newcommand{\norm}[1]{\| #1 \|}\) \( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\) \( \newcommand{\Span}{\mathrm{span}}\)\(\newcommand{\AA}{\unicode[.8,0]{x212B}}\)
Footprinting is a method for determining the exact DNA sequence to which a particular DNA-binding protein binds. Examples:
- 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
- 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.