Question

6. A mutation in one gene knocks out transcription of most mRNA genes in eukaryotic cells, what does this gene encode?

7. If you were to study where P53 regulates an oncogene, you will begin your study by examining whether P53 binds to the promoter of the oncogene; a. Briefly describe the role of transcription factor P53. b. Which methods can be used for your study and what are the expected results?

Answer 1

6- The most probable explanation is that gene encodes for a transcription factor which results in the expression of many genes. This transcription factor binds to the promoter of the gene which it regulates.

7 -p53 is a transcription factor which gets activated upon DNA damage. Upon activation, it leads to the expression of many proteins such as p21, BAX, PUMA, NOXA, MDM2, GADD45 and many other genes.

p53 has a DNA binding domain which helps it to bind to the promoter of the gene and regulates their expression.

We can check the p53 transcription activity by using Electrophoretic Mobility Shift Assay (EMSA) and luciferase assays.

For EMSA we need to design a primer which has a p53 binding site in it. Upon activation levels of p53 increase in the cell. When we incubate the cell lyase with the primers, p53 will bind to its binding site on the DNA. If p53 is activated then we will get the pattern as shown in the below figure.

In luciferase assay, we clone the promoter in the plasmid which the luciferase gene under the promoter of the gene which is regulated by p53. Upon activation levels of p53 is increased which leads to its binding the luciferase plasmid. As the p53 bins to the promoter, it will start expressing the luciferase gene.

Levels of luciferase can be measured by luciferase assay.

if p53 is activated then we will get more amount of luciferase activity