GAL4 derivatives function alone and synergistically with mammalian activators in vitro

We show that derivatives of the yeast transcriptional activator GAL4, synthesized in and purified from E. coli, stimulate transcription of a mammalian gene (the adenovirus E4 gene) in a HeLa cell nuclear extract. Stimulation depended upon GAL4 binding sites inserted in the template. When the GAL4 sites were placed immediately upstream of the E4 TATA box, GAL4 stimulated efficiently. When the GAL4 sites were further upstream from TATA, however, efficient stimulation by GAL4 required, in addition, a site for a mammalian transcriptional activator immediately upstream of TATA. Under these conditions, the GAL4 derivatives functioned synergistically with the proximally bound activator. Previous experiments have defined two "activating regions" in GAL4, and our current experiments define a third, whose function is observed in vitro but not in vivo.