Question

Why does the engineered protein no longer regulate transcription of the GAL genes? How might you alter the DNA-binding site recognized by the new chimeric protein to rescue it to make it functional in activating transcription of GAL genes?

Answer 1

Engineered protein is the chimeric protein. That means fusion of two proteins. Regulator molecule of GAL operon is Gal 3p. It has activation domain and binding domain. Binding domain binds to the UAS(G) sequences of operon and regulate transcription in responce to the galactose availability. Engineered chimera protein losses its ability to bind to the UAS(G) sequences . Therefore, these chimera protein must have been engineered in the UAS(G) binding domain. Therefore it is not able to recognize UAS(G) and hence could not regulate transcription.

To rescue it. You must have to identify the DNA binding sequences of the engineered domain of the chimera protein. Following steps can be perform to know it

1. Generate the synthetic DNA sequences varying few nucleotides of the UAS(G)

2. Check for the binding affinity for these synthetic DNA with the engineered chimera protein.

3. Select all those sequences which have average and above binding affinity with the engineered protein.

4. Crystalize each protein with its counter DNA. If protein is difficult to crystalize the use NMR to solve the structure.

5. Perform structural alignment for each structure by using bioinformatics software.

6. Perform PWM prediction of the superimposed structure obtained in step 5.

7. You will get the DNA sequence which will binds to your chimera protein.

Insert this DNA sequence in the place of UAS(G). Transcription regulation will be rescued with engineered chimera protein.