CHARACTERIZATION OF THE ELK-1 ETS DNA-BINDING DOMAIN

The ETS domain family of transcription factors is comprised of several important proteins that are involved in controlling key cellular even ts such as proliferation, differentiation, and development, One such p rotein, Elk-1, regulates the activity of the c-fos promoter in respons e to extracellular stimuli. Elk-1 is representative of a subgroup of E TS domain proteins that utilize a bipartite recognition mechanism that is mediated by both protein-DNA and protein-protein interactions, In this study, we have overexpressed, purified, and characterized the ETS DNA-binding domain of Elk-1 (Elk-93), Elk-93 was expressed in Escheri chia coli as a fusion protein with glutathione S-transferase and purif ied to homogeneity from both the soluble and insoluble fractions using a two-column protocol. A combination of CD, NMR, and fluorescence spe ctroscopy demonstrates that Elk-93 represents an independently folded domain of mixed alpha/beta structure in which the three conserved tryp tophans appear to contribute to the hydrophobic core of the protein, M oreover, DNA binding studies demonstrate that Elk-93 binds DNA with bo th high affinity (K-d approximate to 0.85 x 10(-10) M) and specificity , Circular permutation analysis indicates that DNA binding by Elk-93 d oes not induce significant bending of the DNA. Our results are discuss ed with respect to predictive models for the structure of the ETS DNA- binding domain.