Interaction of DNA binding domain of HNF-3 alpha with its transferrin enhancer DNA specific target site.
Transferrin hepato-specific gene enhancer, associated with the liver-enriched HNF-3 alpha transcriptional factor and ubiquitous proteins, is a complex molecular edifice maintained through DNA-protein and protein-protein interactions. As a first step to understand the mechanisms responsible for its organization and activity, we have analyzed the interaction of the DNA binding domain of HNF-3 alpha (HDBD) with a specific DNA segment present in the transferrin enhancer by different biophysical techniques. The kinetic constants of this interaction were measured using surface plasmon resonance. The HDBD-DNA interaction was also characterized by circular dichroism and fluorescence spectroscopy. HDBD binds to its specific DNA site with high affinity (Kd approximately equal to 10(-8) M). The affinity is reduced after sequence modification of the target DNA. Size exclusion chromatography and binding stoichiometry determined by fluorescence measurements indicate that the protein is present in a monomeric form before and after interaction with the DNA. The secondary structure of the protein was not significantly altered upon binding to specific DNA. By contrast, a structural change of DNA by interaction with HDBD seems to occur.
Terenzi
H
H
Unité d'Expression des Gènes Eucaryotes, Institut Pasteur, Paris, France.
Petropoulos
I
I
Ellouze
C
C
Takahashi
M
M
Zakin
M M
MM
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D051541:Hepatocyte Nuclear Factor 3-alpha; D007700:Kinetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014157:Transcription Factors / Q000378:metabolism*; D014168:Transferrin / Q000235:genetics*
D004268:DNA-Binding Proteins; D051541:Hepatocyte Nuclear Factor 3-alpha; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D014168:Transferrin; D004247:DNA