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Two-Hybrid System | imsd016


Two-Hybrid System
The Two-Hybrid System is based on reconstitution of a transcription-factor by protein-protein interactions. Transcription-factors are proteins which bind to DNA and activate genes. Such a transcription-factor consists of two domains, a DNA-binding domain and a activation-domain. Only together they can fulfill their function. If they are seperated from each other, the DNA-binding-domain still remains able to bind to DNA, but since the activation-domain is missing, there is no gene-activation anymore. We can use this fact, because it is possible by genetic engineering to generate fusion-proteins. Such a fusion-protein consists for example of the DNA-binding-domain and a fused protein of interest, e.g. MEKK, and a second fusion-protein would be formed by the activation-domain and a second protein which has to be tested for interaction with MEKK. If both proteins are able to bind to eachother, the transcription-factor is reconstituted and genes are activated. These gene-activations we can measure in our experiments and the results let us know whether the tested factors are able to interact with each other. By generating of many such fusion-proteins - a so called library - which also might contain so far unknown factors, and by testing all of them with MEKK, we can detect, which interactions occur with MEKK. We also can identify new, unknown interaction-partner. This identification of course is only the first step. It is followed by the discovery of the function and the meaning of this interaction for the cell. That is the difficult part of the research, which is very frustrating, but which also is connected with a lot of joy.


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