INTERACTION OF DNA-BINDING DOMAIN OF HNF-3-ALPHA WITH ITS TRANSFERRINENHANCER DNA SPECIFIC TARGET SITE

Transferrin hepato-specific gene enhancer, associated with the liver-e nriched HNF-3 alpha transcriptional factor and ubiquitous proteins, is a complex molecular edifice maintained through DNA-protein and protei n-protein interactions. As a first step to understand the mechanisms r esponsible for its organization and activity, we have analyzed the int eraction of the DNA binding domain of HNF-3 alpha (HDBD) with a specif ic DNA segment present in the transferrin enhancer by different biophy sical techniques. The kinetic constants of this interaction were measu red using surface plasmon resonance. The HDBD-DNA interaction was also characterized by circular dichroism and fluorescence spectroscopy. HD BD binds to its specific DNA site with high affinity (K-d congruent to 10(-8) M). The affinity is reduced after sequence modification of the target DNA. Size exclusion chromatography and binding stoichiometry d etermined by fluorescence measurements indicate that the protein is pr esent in a monomeric form before and after interaction with the DNA. T he secondary structure of the protein was not significantly altered up on binding to specific DNA. By contrast, a structural change of DNA by interaction with HDBD seems to occur.