Abstract: Several transcriptional activators, called "classical" because each bears a natural acidic activating region attached to a DNA binding domain, are proteolytically unstable in yeast, and it has been suggested that this instability is required for transcriptional activation [1-3]. Here we test the generality of that proposal by examining a set of activators (called "nonclassical") that lack activating regions. These activators (e.g., LexA-Gal11) comprise a LexA DNA binding domain fused to a component of the Mediator and are believed to insert the latter into the Mediator and recruit it (and, indirectly, other components required for transcription) to a gene bearing LexA sites [4-8]. We find that three, and only three, Mediator subunits, all from its tail domain, work as activators when fused to LexA. All three are unstable, and for the case analyzed in detail, stabilization decreases activity. Thus, to the extent tested, both classical and nonclassical activators work most efficiently when proteolytically unstable.CI - Copyright (c) 2010 Elsevier Ltd. All rights reserved.