In the electrophoretic mobility shift assay (EMSA), fragments of DNA are incubat
ID: 3166719 • Letter: I
Question
In the electrophoretic mobility shift assay (EMSA), fragments of DNA are incubated with a protein of interest or protein extract containing many proteins and then analyzed on a nondenaturing polyacrylamide or agarose gel The DNA used in the experiment is visualized either by staining with a dye or by covalently attaching a radioactive phosphate group at one end. Free DNA fragments migrate more quickly through the gel than DNA bound by protein. Thus, a shift in migration of DNA from fast to slow in the presence of protein indicates a direct binding interaction between a protein(s) and DNA You are interested in determining which of four proteins, A, B, C, and D, may compete with CAMP-CAP for binding to the lac operon CAP-binding site using EMSA. You mix radiolabeled DNA fragments containing the CAP-binding site sequence with each of the four proteins and perform nondenaturing polyacrylamide gel electrophoresis and autoradiography. The results are shown in Figure1 2 3 4 5 6 Figure 1. EMSA with radiolabeled CAP-binding site DNA fragment. Lane 1, control with no protein added. Lane 2, positive control with cAMP-CAP protein added. Lane 3, protein A added. Lane 4, protein B added. Lane 5 protein C added. Lane 6, protein D added Which protein(s), A, B, C, and/or D appear to bind to the CAP-binding site? A) Proteins B and C B Proteins A and D CProtein A only DProtein B only Which of the following is the most likely explanation for the band pattern seen in lane 6. A) Protein D is a restriction enzyme and cut the DNA fragment into two DNA fragments. B) Protein D binding to the CAP-binding site is less tight compared to cAMP-CAP binding C) Protein D has a smaller mass than the cAMP-CAP protein DProtein D removed radioactive label from the DNA fragment.Explanation / Answer
This question is based on DNA footprinting.
Which protein(s) A,B,C and/ or D appear to bind to the CAP-binding site
The correct answer is option B - Protein A and D.
Reason: The migration of DNA in lane 3 is reduced. This is due to the binding of protein to the CAP binding site. In Lane 6, we can also observe a decline in the migration of DNA. Hence the correct answe is Protein A and D.
Which of the following is the most likely explanation for the band pattern seen in lane 6
The correct answer is option B - Protein D binding to the CAP-binding site is less tight compared to the cAMP-CAP binding.
The loose binding of the protein to the DNA, allows the DNA to migrate faster in gell electrophoresis.