Question 4 You have decided to study the role of SWI1 and SWI2 proteins in trans

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Question 4

You have decided to study the role of SWI1 and SWI2 proteins in transcriptional enhancement via steroid receptors. You know that when a cell is exposed to glucocorticoids (steroid hormones) the intracellular binding of this hormone to its cognate receptor results in the transcriptional activation of a specific sub-set of genes. A promoter called the glucocorticoid response element characterizes these genes.

Yeast cells are co-transformed with the following constructs:

- A yeast expression plasmid, pE, responsible for the constitutive expression of the gene encoding the glucocorticoid receptor.
- A yeast reporter plasmid, pR, containing the gene encoding b-galactosidase under the control of the glucocorticoid response element.

a) Describe how the synthesis of b-galactosidase in the yeast cells is affected by the addition of glucocorticoid. Include in your description the variation in the level of transcriptional activity of the glucocorticoid receptor gene in both the absence and presence of hormone.

b) The activity of b-galactosidase before and after addition of glucocorticoid was measured using three different strains of yeast each co-transformed with pE and pR. The strains are the following:

- SWI+, is a wild-type strain producing active SWI1 and SWI2 proteins

- swi1- and swi2- are two mutant strains producing inactive SWI1 and SWI2 proteins respectively. The results of the b-galactosidase activity measurements were the following:

Based on the fact that proteins SWI1 and SWI2 are found in a multimolecular transcriptional complex, these results.

c) If strains SWI+, swi1- and swi2- are only transformed by the yeast expression vector pE in which the b- galactosidase gene has replaced the glucocorticoid receptor gene the level of b-galactosidase activity is similar in each strain. Why is this control experiment critical to explaining the results presented in part (b)?

Explanation / Answer

a)   the synthesis of b-galactosidase in the yeast cells is get enhanced by the addition of glucocorticoid. The glucocorticoid receptor (GR) signal transduction and transcriptional regulation are efficiently recapitulated when GR is expressed in yeast cell.Glucocorticoid receptor expressed in yeast is phosphorylated at T171, S224, S232, and S246.In the absence of hormone, the inactive apo-GR resides in the cytoplasm in association with molecular chaperones that maintain the aporeceptor in a high-affinity hormone-binding state.

b).the level of b-galactosidase activity is similar in each strain because of similar potential

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