STRUCTURAL INFERENCES OF THE ETS1 DNA-BINDING DOMAIN DETERMINED BY MUTATIONAL ANALYSIS

The ets family of transcription factors is characterized by a conserve d region that harbors the DNA-binding activity. We performed extensive deletion and mutational analyses, as well as DNA-peptide interaction studies necessary to identify the determinants of the DNA-binding acti vity of the ETS1 oncoprotein. We found that amino acids beyond the 85 amino acid conserved region are required in order to afford maximum DN A-binding activity in a heterologous system. Mutation throughout the b inding domain can have a detrimental effect on binding activity, indic ating that proper folding of the entire domain is necessary for DNA bi nding. A peptide, as small as 37 residues (K37N), derived from the bas ic region of the ETS1 binding domain, is sufficient to exhibit sequenc e-specific DNA recognition. Total randomization of Lysine 379, Lysine 381 and Arginine 391 within this region fails to provide functional su bstitutions, indicating that these specific amino acids within the bas ic region are required for binding. Transactivation activity of the ET S1 proteins bearing mutations was consistent with their DNA-binding ac tivity, indicating that the primary (if not only) function of this dom ain is to provide sequence-specific DNA recognition activity. Our muta tional analysis, as well as modeling predictions, lead us to propose a helix-turn-helix structure for the basic region of the ETS1 binding d omain that is able to interact directly with DNA. We also propose that the hydrophobic alpha-helical region, surrounding tryptophan 338, is fundamental for proper protein folding and;functioning of the ets bind ing domain.