破天一剑领孩子:Foot printing

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Footprinting

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 [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.
Link to discussion of DNA sequencing.Link to a view of the actual sequence of the lac operatorLink to a discussion of how transcription factors can be isolated from the cell.Link to a discussion of chromatin immunoprecipitation (ChIP) — another method by which the DNA sequence to which a particular DNA-binding protein binds can be determined.Welcome&Next Search
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