OR contains two promoters. RNA polymerase will readily bind to PR and initiate transcription in a rightwards direction through the cro gene. RNA polymerase may, under the right circumstances, bind to PRM and initiate transcription in a leftwards direction through the cI gene. The ability of RNA polymerase to bind to PR or PRM and to initiate transcription depends upon the presence and the concentration of CI and Cro.

[P1.4]

 

Measuring the Properties of a DNA Regulatory Element

In a very general sense, we need the following items in order to study the regulatory properties of any particular regulatory element in vivo.

 

REGULATORY ELEMENT

It almost goes without saying that we need the regulatory element whose activity we wish to measure. In general this will be a short DNA fragment containing a promoter and some combination of operators or activator binding sites. It is the influence on transcription of proteins bound to the operators or activator binding sites that we wish to examine.

 

REPORTER GENE

If we are going to measure regulation, we must use a gene whose product has an activity that is easy to measure. Common reporter genes are:

lacZ -- b galactosidase

cat -- chloramphenicol acetyl transferase

lux -- luciferase

GFP-- Green Fluorescence Protein

 

A SUPPLY OF THE REGULATOR PROTEIN

It is usually easy enough to supply the regulator protein if the cells contain the gene coding for the protein.

 

A WAY TO REGULATE THE SUPPLY OF THE REGULATOR PROTEIN

Depending on the circumstances, we may wish to be able to regulate the supply of the regulator protein. For example, we may wish to turn the supply on or off. Or we may wish to vary the amount of the regulator protein that is present in the cell to see how the cell responds to the changing concentrations of the regulator.

Thus, we may wish to clone the regulator protein gene under the control of (for example) an inducible promoter.

 

Putting all these elements all together, we obtain a system such as the following:

 

 

If expression is induced from the inducible promoter

 

• then the regulatory protein will be synthesized
  
  
  • it will bind to its binding sites in the regulated promoter segment
    
    
    • which will affect the level of expression of the reporter gene
      
      
      • which can be measured by assaying the amount of reporter gene product that is present.

 

Similarly, if the level of expression from the inducible promoter is modulated by the addition of an inducer

 

• then the amount of regulatory protein synthesized will change
  
  
  • which will change the amount of the regulator protein bound to the binding sites in the regulated promoter segment
    
    
    • which will affect the level of expression of the reporter gene
      
      
      • which can be measured by assaying the amount of reporter gene product that is present.
        

 

Control of Transcription at OR by Repressor Control of Transcription at OR by Cro