CHARACTERIZATION OF THE COOPERATIVE FUNCTION OF INHIBITORY SEQUENCES IN ETS-1

DNA binding by the eukaryotic transcription factor Ets-l is negatively regulated by an intramolecular mechanism, Quantitative binding assays compared the DNA-binding activities of native Ets-l, three deletion m utants, and three tryptic fragments, Ets-l and activated Ets-l polypep tides differed in DNA-binding affinity as much as 23-fold, Inhibition was mediated by two regions flanking the minimal DNA-binding domain, B oth regions regulated affinity by enhancing dissociation of the protei n-DNA complex, Three lines of evidence indicated that inhibition requi res cooperative interaction between the two regions: first, the two in hibitory regions acted through a common mechanism; second, neither reg ion functioned independently of the other; finally, mutation of the C- terminal inhibitory region altered the conformation of the N-terminal inhibitory region. In addition, partial proteolysis detected an identi cal altered conformation in the N-terminal inhibitory region of Ets-l bound to DNA, This finding suggested that repression is transiently di srupted during DNA binding, These results provide evidence that the tw o inhibitory regions of Ets-l are structurally, as well as functionall y, coupled. In addition, conformational change is shown to be a critic al component of the inhibition mechanism, A cooperative, allosteric mo del of autoinhibition is described. Autoinhibition of Ets-l could be r elieved by either protein partner(s) or posttranslational modification s.