Question

12. You have cloned an origin of replication and are interested in identifying the proteins that bring about initiation of replication. You find that proteins in a cell extract bind to your origin. You fractionate the extract and identify three fractions, X, Y and Z, that you believe contain proteins that directly or indirectly bind to your origin. You carry out an EMSA experiment and obtain the data shown in the figure below.

1 2 3 4 5 6 7 8 0 X Y Z X X Y X Y Z Z Y Z

Which of the following (A-F) is order of binding of your fractions to the origin?

Answer 1

Ans: EMSA(Electrophoretic Mobile Shift Assay) technique is based on the observation that protein–DNA complexes migrate more slowly than free linear DNA fragments when subjected to non-denaturing polyacrylamide or agarose gel electrophoresis.

Now as given in the gel run that in the presence of none of the protein or in the presence of X protein or Y protein or x and y protein together the band is forming at the same position (at the farthest position) which means that neither X nor Y protein is directly binding with fraction of origin. So this means that Z is directly binding with the fraction of origin and we can see thta in the gel run that in 4th column the fraction has travelled less as compared to the 0th, 1st, 2nd, 3rd and 5th column.

Now among 6th and 7th column the band in 6th column with X and Z protein has travelled less as compared to the 7th column with X and Z protein which means that Z has more affinity for X protein than for Y protein.

So this means that the order of binding of the proteins to the fraction of origin is : Z directly binds to the fraction and X binds with Z protein and lastly Y binds to the X protein.

Hence option C is correct.