Please Help!! Promoter Bashing Case Study Yeast are unicellular eukaryotic fungi
ID: 205908 • Letter: P
Question
Please Help!!
Promoter Bashing Case Study Yeast are unicellular eukaryotic fungi often used in biological research because they are cheap and easy to manipulate genetically. The LacZ gene encodes a protein named -galactosidase, which converts lactose into galactose and glucose. We can use the lacZ gene as a reporter gene to analyze reporter functions. In this case, lacZ is placed in the DNA downstream of the promoter that regulates TBP (TATA-binding protein) takaryotie Promoter DNA seqaences ect pre ttow Product lysis A420 Researchers have 4 strains of yeast. 1. The E strain contains the lacZ gene, but no promoter ("empty" promoter). 2. The P strain contains the lacZ gene under control of the TBP promoter ("promoter" strain). 3. The A strain and... 4 .the B strain are experimental strains with mutations in the regulatory regions. The gene schematic is shown below. The A and B strains have deletions in their respective areas shown below. promoter lacz geneExplanation / Answer
The answer to question 12
The "E" strain is showing low-level of beta-gal activity because:
a. The E strain has lacZ gene but no promoter.
b. The promoter region is very much essential for the binding of RNA polymerase.
c. The expression of lacZ is dependent on the binding of RNA polymerase to the promoter region
d. Due to the absence of promoter region, lacZ is not expressed and hence beta-gal activity is less in E strain.
The answer to question 13
a. The region that is deleted in the strain A is neither an enhancer nor an operator.
An enhancer increases the expression of lacZ. The deletion of enhancer would have reduced the beta-gal activity. But from the given table, the activity is high in this strain. So, the deleted region is not an enhancer.
In lac operon, an operator is positioned after the promoter. In the given figure we can observe that the deletion has been shown far away from the promoter of lacZ gene. As per the lac operon, the deleted region in strain A corresponds to the lac repressor gene lacI. Due to this deletion, the gene lacI failed to express lac repressor protein and thus there was no repression of lacZ gene. As a result, we can observe an increase in beta-gal activity in strain A.
b. The region that is deleted in the strain B is an enhancer. If we closely observe the given figure, the deletion of regulatory region in strain B is very close to the promoter. This region has TATA box which acts as enhancers of transcription. TATA-binding protein specifically binds to TATA box and augments the rate of transcription. The deletion of this region reduced the rate of transcription and thus we can observe very low beta-gal activity in strain B.
The answer to question 14
If amatoxin is added one hour prior to ONPG addition, then the strain P would have shown very less enzyme activity. The expression of lacZ stops and the beta-galactosidase is not secreted by the yeast.
This is because amatoxin specifically binds to bridge helix region of RNA polymerase II and blocks the translocation of DNA into the RNA polymerase. As a result, mRNA is not synthesized and the gene fails to express the desired protein.
Strain P is a promoter strain. RNA polymerase II specifically binds to the promoter region. Addition of amatoxin inhibits the activity of RNA polymerase II. So, we can predict that the strain P will not be able to express beta-galactosidase.