Question

Microbiology: You have the following working hypothesis: To bind well to a DNA-binding protein, a DNA target site must twist less tightly and widen the narrow groove between base pairs 4 and 5. Suggest an experiment to test your hypothesis.

Answer 1

The structure of Deoxyribonucleic acid (DNA) has a highly conserved stoichometry. It has two strands which run in an antiparallel manner, and forming a helical structure due to base pairing between nucleotides. The regulation of many important biological processes involve binding of DNA with proteins, known as the DNA-binding proteins. For efficient binding, the DNA microstructure is such that the target site for binding is located majorly at the major groove of the DNA helix (mainly between base pairs 4 and 5). In the major groove, there is a 'kink' or bend of about eighty degrees for binding of TBP or TATA-box binding proteins, which are the most important DNA-binding proteins. This kink or bend helps in increasing the surface area and providing a platform for easy binding of proteins to the DNA microstructure.

For proving this hypothesis, we can detect the changes in the DNA microstructure before and after the binding of DNA-binding proteins. We can also rely on the techniques used to analyze the DNA and protein interaction. It can be done by the application of proteomics, for example, with the aid of Electron Spray Ionization- Mass Spectrometry. ( ESI-MS). In ESI-MS, the affinity of the DNA binding protein to the actual target site on the major groove with the bend can be analyzed and compared with the non-target site binding. Other techniques like DNA footprinting, Chromatin Immunoassay precipitation (ChIP) supershift, Southwestern blotting can also be used.