Abstract 14071: Mechanisms of Rapid Action by Stress-inducible Transcription Factor Egr-1
The cardiovascular system should quickly and appropriately respond to stresses that can pose a threat to health. The stress-inducible transcription factor Egr-1 is induced in endothelial cells by vascular stresses, and plays a key role in the response to environmental challenge to the cardiovascular system. By binding to 9-bp target DNA sites via three zinc-finger domains, the induced Egr-1 protein rapidly activates genes for defense against the stresses. Our goal is to elucidate the mechanisms allowing Egr-1 to accomplish both rapid target search and stable target association. Extensive contact with DNA via the three zinc fingers can enhance stability of the complex with the target, but can also slow the search process because a larger number of interactions with DNA must be broken whenever protein transfers from one nonspecific site to another. In our current biophysical study, we assessed the hypothesis that Egr-1’s domain motions play an important role in DNA-scanning. We modulated Egr-1’s domain motions in DNA scanning via rational mutagenesis, and studied how the modulation influences kinetics and thermodynamics of target association. Using NMR spectroscopy, we studied the impact of the mutations on the protein structure and dynamics in DNA-scanning. Using fluorescence spectroscopy, we investigated the kinetics of the target search process and thermodynamics of specific and nonspecific protein-DNA interactions. Our data indicate that Egr-1 efficiently scans DNA via dynamic transitions between two structurally different states, which are termed the search mode and the recognition mode. In the search mode, a zinc finger is dissociated from DNA while the other two zinc fingers are nonspecifically bound to DNA. The search mode allows rapid translocation of Egr-1 on DNA, whereas the recognition mode allows highly specific recognition of the target DNA sequence. Our study provides new insights into how the stress-inducible transcription factor Egr-1 can rapidly regulate target genes in the response to the vascular stresses. This work was supported by Grant 12BGIA8960032 from the American Heart Association (to J.I.).
- © 2013 by American Heart Association, Inc.