Artificial recruitment has been demonstrated in vitro and in vivo (refs. It is believed to insert its Srb2 moiety into the holoenzyme binding to a Gal4 site then recruits the holoenzyme to the nearby promoter (see ref. Gal4 + Srb2, an example of such a fusion protein, bears the holoenzyme component Srb2 fused to the Gal4 DNA-binding domain. In an alternative strategy, called “artificial recruitment,” the machinery is brought to the promoter either by an arbitrary protein–protein interaction between the machinery and a DNA-tethered peptide or by the action of a fusion protein comprising a DNA-binding domain and a component of the transcriptional machinery ( 5, 6). One way to demonstrate this effect in vitro is by using high concentrations of bacterial RNA polymerase or of the yeast RNA polymerase II holoenzyme plus auxiliary factors ( 2, 3). These experiments show that the effect of an activator can be dispensed with-and the activator has no further effect-provided that that machinery can be brought to the promoter by some other means. So-called “activator-bypass” experiments, performed in yeast and bacteria, have played an important role in formulating the idea that many transcriptional activators work by recruiting the transcriptional machinery to the promoter ( 1– 4). The results, consistent with those of analogous experiments in yeast, suggest that classical activators, presumably by virtue of their abilities to interact with multiple targets, have a functional flexibility that nonclassical activators lack. The effect of the one nonclassical activator that does elicit significant levels of transcription when working alone (i.e., that bearing TATA box-binding protein) is strongly influenced by promoter architecture. The effect of the classical activator in such experiments requires that it be tethered to DNA, a requirement that cannot be overcome by expression of that classical activator at high levels. However, as found in analogous “artificial recruitment” experiments performed in yeast, these activators work synergistically with “classical” activators.

We show that the typical “nonclassical” activator, which comprises a fusion protein bearing a component of the transcriptional machinery fused to a DNA-binding domain, activates transcription in mammalian cells only weakly when tested with an array of promoters.

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