The strength of acidic activation domains correlates with their affinity for both transcriptional and non-transcriptional proteins

Activation domains (ADs) appear to work by making specific protein-protein contacts with the transcriptional machinery. However, ADs show no apparent sequence conservation, they can be functionally replaced by a number of ran dom peptides and unrelated proteins, and their function does not depend on sustaining a complex tertiary structure. To gain a broader perspective on t he nature of interactions between acidic ADs and several of their proposed targets, the in vivo strengths of viral, human, yeast, and artificial activ ation domains were determined under physiological conditions, and mutant AD s with increased iir vivo potencies were selected. The affinities between A Ds and proposed targets were determined in vitro and all interactions were found to be of low-level affinity with dissociation constants above 10(-7) M. However, iii vivo potencies of all ADs correlated nearly perfectly with their affinities for transcriptional proteins. Surprisingly, the weak inter actions of the different ADs with at least two non-transcriptional proteins show the same rank order of binding and AD mutants selected for increased in vivo strength also have increased affinities to non-transcriptional prot eins. Based on these results, isolated acidic ADs can bind with relatively low-level specificity and affinity to many different proteins and the stren gth of these semi-specific interactions determine the strength of an AD. I suggest that ADs expose flexible hydrophobic elements in an aqueous environ ment to contact hydrophobic patches over short distances, shifting specific ity of activators largely to the DNA colocalization of arrays of ADs and ta rgets. (C) 2000 Academic Press.