You have purified a protein from a bacterium Y that causes cells in tissue cultu
ID: 78357 • Letter: Y
Question
You have purified a protein from a bacterium Y that causes cells in tissue culture to increase their cAMP levels. 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 transcriptional 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.
Cyclohexamide is a protein synthesis inhibitor.
So, grow cells in a media that does not contain Fe and add cyclohexamide to it. If the iron deficient conditions induce transcriptional activation of Yct A, we can quantify its levels using PCR. The addition of cyclohexamide prevents the protein synthesis from already existing mRNAs in the cell.
B.
To check whether the given sequence codes for a transcription factor,
1. Do DNA binding assay - for example SELX to identify which specific sequences it can bind. Any basic protein would bind negatively charged DNA in a nonspecific manner. To negate this possibility, clone the DNA binding sequence of the protein upstream to a reported gene (GUS or GFP) and transform cells along with the protein of interest. If the marker gene shows expression that concludes that the given protein binds to DNA and serves as transcription factor.
C.
It is a very difficult task to decide whether a given transcription factor is an activator or repressor. Most of the times, a single transcription factor can serve as both activator and repressor for different genes. This is achieved by association of accessory proteins or epigenetic machinery.
On a broad scale, if we want conclude whether a transcription factor acts as an activator or repressor, we can perform a microarray in the presence of cyclohexamide for the given transcription factor induction. If we get more number of genes unregulated in the micro array than repressed genes, it shows that the transcription factor could be an activator. But, this is an indirect way.
If we know the target gene of transcription factor, we can clone its promoter region upstream to a reported gene and check whether transcription factor induction can activate or repress gene expression.
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