(Solved): 3. You go on to sequence the Pacl gene in an additional patient (Patient 4) and identify a variant t ...

3. You go on to sequence the Pacl gene in an additional patient (Patient 4) and identify a variant that you believe will disrupt the DNA binding domain of the PAC1 protein. In order to test your hypothesis, you amplify radiolabeled segments of DNA representing the -60 to +1 region of a promoter for the Man2 gene, which contains a PAC1 binding sequence. You then perform an Electrophoretic Mobility Shift Assay by mixing the radiolabeled DNA segment with either wild type PAC1 protein or PAC1 protein generated from DNA bearing your variant of interest. a) Please draw what your gel would look like if your hypothesis is correct and the variant in Patient 4 does indeed disrupt PAC1 binding to the Man2 promoter. (1 mark) b) What would be the effect if you ran your EMSA gel under denaturing conditions? (1 mark) Why? (1 mark) c) What would be the effect of the variant in Patient 4 on the transcription of the Man2 gene (1 mark)? Why (1 mark)? d) PACl does not bind RNA Pol-ll directly, but is instead bound by a protein called PBP, which then interacts with RNA Pol-II. What is PBP in this scenario? (1 mark) e) Please draw what your gel would look like if you were to run an EMSA with the following lanes ( 2 marks) i) Man 2 promoter alone ii) Man2 promoter + wild type PAC1 iii) Man2 promoter + wild type PACl + PBP