3) The gene coding for b-galactosidase is under the control of an inducible prom
ID: 267569 • Letter: 3
Question
3) The gene coding for b-galactosidase is under the control of an inducible promoter. Therefore, the gene is transcribed only when a diffusible inducer is added to the yeast culture.
Experiment 3: The inducer was added to the yeast culture for 2 minutes, and then the cells were harvested and resuspended into a fresh culture medium containing a transcriptional repressor of the b-galactosidase gene. Samples of yeast cells were taken from the culture at different time intervals before or after the addition of the repressor. The b-galactosidase activity in these samples was immediately measured. Alternatively, mRNA was extracted immediately after the addition of the repressor and stored on ice. At different time intervals, these mRNA samples were translated and the b-galactosidase activity in these in vitro translation reactions was measured.
a) Is experiment 3 a pulse-chase experiment? Briefly explain b) The following graphs represent the results of experiment 3.
Describe the two biological properties of the polyA tail illustrated by experiment 3. Briefly explain.
WT WT M2 Time Time of incubation of the purified mRNA before in vitro translation + RepressorExplanation / Answer
Answer a)
The experiment is not a pulse chase experiment becuase neither nucleotides ( present in mRNA ) nor amino acids ( present in beta-galactosidase enzyme ) are radiolabelled/flourescent labelled. The labelled biomolecules (nucleotides/amino acids ) are incorporated into macromolecules (nucleic acids or proteins) and are used to analyse the effect of various compounds/molecules on the cellular processes like DNA transcription or proteins translation. In the present experiment, none of radiolabelled aminoc acid of nucleotides has been used, only an unlabelled inducer/repressor has been added to the growing yeast cells.
Answer b)
The addition of repressor gradually reduces the level of transcription of mRNA for beta-galactosidase enzyme. Thus we see a an increasing trend. As the time of incubation increases the repressor diffuses into the yeast cells and slowly reduced the transcription of mRNA for beta galactosidase by binding to the operator region of promoter for beta galactosidase.
In vitro translation decrases becasue the mRNAs were immediately harvested after transcription. Hence they were unable to complete poly A tail addition to their 3'-ends. As the time of incubation elapsed, they were being subjected to dgradation, showed a steady reduction in translation.
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