You further investigate the regulatory proteins that bind to Regions 1, 2 and 3

Question

You further investigate the regulatory proteins that bind to Regions 1, 2 and 3 using gel shift assays. A gel shift assay uses labeled DNA as a target for regulatory protein binding.

A labeled DNA fragment that fails to bind a protein will migrate quickly on an electrophoretic gel.

A labeled DNA fragment that does bind a protein will migrate more slowly because the DNA + protein complex is larger than the DNA alone. Thus, the label associated with the DNA will “shift” to a higher location on the gel, as shown in this diagram.

To investigate regulatory protein binding to Regions 1, 2, and 3, you will use labeled DNA fragments with some of the same point mutations as in Part B, incubate them with proteins extracted from heart or lung tissue, and conduct a gel shift assay. How would gel shift assay results be affected by these mutations?

Select the two correct answers.

1. Region 2 binds protein from lung tissue. If Region 2 is mutated, then the DNA will not shift on the gel when incubated with protein from lung tissue. 2. Regions 1 and 3 bind protein from heart tissue. If both Regions 1 and 3 are mutated, then the DNA will not shift on the gel when incubated with protein from heart tissue. 3. Region 2 binds protein from heart tissue. If Region 2 is mutated, then the DNA will not shift on the gel when incubated with protein from heart tissue. 4. Regions 1 and 3 bind protein from lung tissue. If both Regions 1 and 3 are mutated, then the DNA will not shift on the gel when incubated with protein from lung tissue.

Explanation / Answer

Electrophoretic mobility shift assay (EMSA) or mobility shift electrophoresis:

DNA contains specific sequences to bind with protein. If DNA will bind with protein its molecular weight will increase and its will shift its position in the gel. But if DNA will be mutated, the specific protein will not be able to bind with DNA and therefore DNA will be taking same position in the gel according to its molecular weight. As molecular weight of DNA will remain same as protein is not binding.

As per the data given in the question: all the following given options are correct (about Part B is not mentioned in question):

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