1. You have purified a protein from a bacterium Y that causes cells in tissue cu
ID: 253969 • Letter: 1
Question
1. You have purified a protein from a bacterium Y that causes cells in tissue culture to increase their cAMP levles. You subsequently cloned a fragment of DNA from bacterium Y that contained two ORFs, one of which you identified as the gene encoding the protein you isolated, which you called yctA (for Y bacterium cytotoxin A). You then determined that expression of the YctA protein occurs only under iron- restrictive conditions (i.e., when iron is absent from the medium).
A. Describe how you would determine if the expression of YctA is regulated by iron at the transcription level.
B. Describe how you would determine whether the other ORF, which you have named yctR, encodes a possible transcriptional regulator.
C. If YctR is a transcriptional regulator, how could you determine if it is an activator or repressor protein?
Explanation / Answer
A. To determine whether the expression of the given gene is under the control of Iron, grow cells in the presence and absence of iron. Measure the transcript levels in both conditions. If iron is required for the induction of the gene expression, we would observe elevated transcript levels in the qPCR experiment. If iron has no effect, we would not observe any change in transcript level. If iron inhibits the gene expression, we would observe reduced accumulation of transcript in the presence of iron.
B. To determine whether YctR is a potential transcription regulator, perform EMSA. This will tell whether the given protein is able to bind DNA directly which is a prerequisite for a transcription factor. We can also perform ChIP experiment or yeast 1 hybrid experiment.
C. If YctR is a positive regulator of transcription,
i. In the mutant background of YctR, the transcript level of the gene of interest would be decreased. In the overexpression line of YctR, the transcript level of the gene of interest would be increased.
If YctR is a negative regulator of transcription,
i. In the mutant background of YctR, the transcript level of the gene of interest would be increased. In the overexpression line of YctR, the transcript level of the gene of interest would be decreased.
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