The Lactose Operator

Biochemical characterization of the lactose operator and its interaction with repressor has involved:

• isolation of the operator-containing DNA.
  
  
• nucleotide sequencing of the operator.
  
  
• determination of the sequence changes in O^C mutations.
  
  
• DNase footprinting analysis.
  
  
• Methylation protection and ethylation protection experiments.
  
  
• Analysis of specific interactions in Repressor-Operator co-crystals.

 

The characterization of the lactose operator(s) has served as an example for the characterization of many other operators.

 

Isolation of the Operator

The isolation of the operator was based on:

• nitrocellulose filter-binding of protein and protein-DNA complexes
  
  
• nuclease digestion of protein-DNA complexes to remove all unprotected DNA

 

The following steps outline the procedure:

• Bind repressor to a DNA fragment containing the operator.
  
  
• Digest the DNA-repressor complex completely with DNase I or Exonuclease III.
  
  
• Isolate the DNA-repressor complexes by nitrocellulose filter-binding.
  
  
• Recover the undigested DNA from the bound complexes.
  
  
• Sequence the DNA to determine the extent of the operator and to identify sequence elements.
  

 

In the case of the lactose operator, a 26 bp fragment, extending from -5 to +21 with respect to the startpoint of transcription, was identified by this technique. Analysis of this fragment, revealed that it was a subset of a larger palindromic region that extended from -7 to +28. Palindromic sequences are often found in protein binding sites and frequently indicate that the protein binds as a dimer. Further proof that the sequence was indeed the operator was provided when it was found that O^C mutations mapped within this region and when the individual base pair changes were determined.

[11.7] 12-6

 

The extent of the DNase I footprint on each strand is shown by the pink shading; the yellow boxes indicate symmetrical sequences; the horizontal arrow indicates the startpoint of transcription.

 

Within the same region, DNase footprinting and chemical protection (only methylation data are shown above) experiments support the importance of this region and its role in binding repressor.

Examples of:

• DNAse I footprinting and kinetic analysis
• Ethylation protection and hydroxyl radical analysis
• Methylation protection and gel shift