5 marks 2aDesign an experiment to find out how a protein Xwh

5 marks

2.(a)Design an experiment to find out how a protein X,which you suspect is a transcription factor,binds to an enhancer element of a gene and also interacts with the RNA transcription machinery to activate transcription.You have done a structural analysis of X and have found that it is prdicted to have a domain that binds to DNA and another domain which binds to protein.

(b)Why other cellular proteins might interact with protein X?

Solution

Answer:

2. (a)  Previous studies have suggested that transcription factors mediate the interactions between enhancers and promoters / transcription machinery. Chromatin Immunoprecipitation (ChIP) is a immunoprecipitation technique through which we can determine the interaction of proteins (transcription factors) on promoters or other binding sites such as enhancers.

The ChIP assay involves the following steps:

i. DNA and associated proteins in chromatin from living cells are crosslinked.

ii. The DNA-protein complexes are then subjected to sonication or nuclease digestion into 500 bp fragments.

iii. Crosslinked DNA-protein complexes are precipitated down using specific antibody.

iv. The DNA associated fragments associated with the protein are purified and sequenced to reveal the identity.

If the transcription factor binds to the enhancer DNA and promoter, ChIP analysis can reveal the sequences of the individual DNA fragments.

If the transcription factor binds to enhancer DNA and a protein of the transcription machinery (say RNAP), a protein-protein pull down experiment can be done to identify the protein in 2D gel. The protein band can be sliced out and MS-MS (Mass Spectrocopy) done to delineate the sequence of the bound protein.

Moreover, the exact orientation of the transcription factor binding to the whole comple (enhancer and transcription machinery) can be identified using high resolution Cryo-EM structure determination (X-ray crystallography may be tough in this case).

(b) The protein X already has a domain which can bind to proteins. The same domain can be used by the protein to interact with other proteins through different or overlapping surfaces. Such cases have been seen in proteins containing SH2 and SH3 domains. The protein may also use a different surface (with specific residues) for interaction.

Another possibility may be a change in conformation of protein X upon binding to the transcription machinery. This might open up interacting residues which otherwise may be hidden within a pocket in the protein X.