Explain how the subunits of RNA polymerase II of yeast were

Explain how the subunits of RNA polymerase II of yeast were identified using epitope tagging.

Solution

The RNA polymerase II of yeast and humans is responsible for the transcription of DNA for the synthesis of mRNA which further gets translated to proteins. The purified RNA Pol II has 10-12 and is incapable of recognizing promoter. Epitope tagging is a technique which uses a known epitope and is fused to a recombinant protein by the help of genetic engineering. By choosing an epitope for which the antibody is available it makes the process of detecting proteins possible for which the antibody is not available.

It is difficult to study the polypeptides and co-purify with the polymerase activity and is not possible to say which are subunits and which are contaminants. There are two approaches to the study of the enzyme. First, to separate the subunits and reconstitute the structure and check its function, but it\'s not possible to reconstitute the RNA Polymerase from its subunits. Second, to find the genes for all the subunits and then mutate them and look for its function. The Epitope tagging is the process by which the subunits are identified and studied. The labelled RNA polymerase is epitope tagged and then is immunoprecipitated with the antipope antibody. The anti-epitope binds to the tagged epitope. After this, the use of strong detergent SDS causes the separation of subunits of the RNA polymerase. The subunits are then detected by the process of electrophoresis. Electrophoresis gives distinct bands for the different subunits.