Transactivation Activity

Transactivation is when a gene at one locus stimulates expression of a gene at another locus. This applies to p73 because p73 is a transcription factor of a number of genes (see What is p73?). Expression of p73 results in expression of these genes.

Spacer length affects the ability of p73 to activate transcription. With each increase in the length of the spacer in the response element there was a significant decrease in the activity of p73, to the point where 2bp and 4bp spacers produced basal levels of transcription.

In contrast, p53’s activity did not decrease dramatically, and it was able to tolerate all spacer lengths.

HOW DID THEY DO THIS?

Using a luciferase assay!

The research team engineered a plasmid vector to contain:

                - p73 response elements as an enhancer, upstream to…

                - a minimal promoter, which controlled expression of…

                - the luciferase gene

Figure 14 shows a general map of the plasmid



Figure 14: Map of plasmid used for luciferase assay, showing enhancer, promoter and luciferase gene (Affymetrix Inc., 2012)



The plasmid was inserted into yeast and p73 and luciferin were added. If p73 successfully activated transcription of the luciferase gene, bioluminescence resulted. This is because luciferase catalyses luciferin into oxyluciferin (inactive) and light (see Figure 15):

Figure 15: Reaction catalysed by luciferase (lifesci.ucsb.edu, 2012)

The level of bioluminescence was measured, which showed the level of activity of p73 transactivation: more bioluminescence meant a higher level of p73 activity.