Question

300 Full length protein Mutant protein 1 Mutant protein 2 75 300 1 250 Mutant protein 3 (fig69) 300 100 1 225 Mutant protein 4 Mutant protein 5 100 225 No protein Full length protein Mutant protein 1 Mutant protein 2 Mutant protein 3 Mutant protein 4 Mutant protein 5 (fig70) - In addition to cloning normal full length MYC, you make a series of mutant versions to identify the key functional domains of the protein. Based on similarity to other proteins, you suspect it binds the DNA sequence CACGTG. You make a reporter construct that contains this motif in a control region adjacent to the promoter and test for reporter expression when expressing the different versions of the protein (top figure). You find only the full length protein and mutant protein 1 activate reporter expression. Separately, you harvest the proteins from bacteria and test to see which versions bind DNA using EMSA (second figure). A) Using these two experiments, identify the regions of amino acids in MYC that correspond with any DNA binding domain, activation domain, or repression domain. (3 pts) B) How could you use the EMSA assay to demonstrate the MYC specifically binds the CACGTG sequence and not the similar sequence CAGGTG? (2 pts)

Answer 1

EMSA is an Electrophoretic Mobility Shift Assay used to study protein-DNA or protein-RNA interactions. The EMSA is a electrophoretic separation of Protein-DNA mixture on gel. Different molecules moves through the gel at different speed according to their size,mass. No protein lane represents the unbound DNA. The mixture poured into this lane donot contain any protein. All other lane poured by the mixture with protein. Only 3 lane produced 2 different bands or shift . Means this three proteins- Full length protein, Mutant1 and Mutant3 contains the DNA binding domain.

A)

Common region between this 3 proteins are 250-300 region. So the DNA binding Domain region of amino acids in MYC is 250-300.

Mutant protein 1 and Full length protein only activates reporter expression. Mutant protein 3 did not activate reporter expression. Common region between mutant1 and full length protein which donot present in mutant3 is 75 to 100 amino acid region.

So the Activation domain in MYC protein is amino acid 75 to 100 region.

B)

We can use radiolabeling to the target sequence. The CACGTG sequence would be Radiolabeled with ​​​​​³²P. So that after electrophoresis , the gel should be exposed to x-ray film to observe the result.

If the radiolabeled sequence seen in the EMSA band shift then we can conclude that MYC specifically binds with CACGTG.