Question

If you were to replace the DNA-binding domain of the Yeast Gal4 protein with the DNA binding domain from the Lac repressor, this protein would no longer regulate the transcription of GAL genes in yeast.

A: Draw two different modular diagrams (just pairs of labeled rectangles of domains) of the different functional domains you would expect to find in the Gal4 protein and in the engineered protein.

B: Why does the engineered protein no longer regulate transcription of the GAL genes? How might you alter the DNA-binding site recognized by the new chimeric protein to rescue it to make it functional in activating transcription of GAL genes?

Answer 1

A:

Ans:

1. Hoe Yeast Gal4 protein regulates GAL gene expression:

This protein is a positive regulator for the gene expression of the galactose-induced genes which code for the enzymes used to convert galactose to glucose.

Gal4 protein:

2. Inhibition of GAL gene expression :

Engineered Gal4 protein:

B.

Ans:

i. For successful expression of Gal genes, the presence of Upstream Activating Sequence Gene (UASG) is required. To this UASG Gal4 protein binds as a homodimer and then it leads to transcription followed by expression of GAL genes. Now when this region was replaced by Lac repressor Gal4 protein will no longer recognize it and hence can not bind, therefore no expression of GAL genes. In other words only Gal4 protein having UASG recognizing domain can bind to UASG and activate GAL gene expression whereas engineered Gal4 which does not possess that leads to repression of GAL gene.

ii. Gal4 binds to UASG, which is closer to GAL gene. Now we can insert UASG domain in between Lac repressor region and GAL genes. In this scenario Gal4 protein recognize UASG even in presence of Lac repressor domain and lead to expression of GAL genes.