Activation of transcription in vitro by recruitment of the yeast RNA polymerase II holoenzyme

Abstract

It has been argued that many transcriptional activators work by "recruitment," that is, by helping the transcriptional machinery bind stably to DNA. We demonstrate here a realization of a strong prediction of this idea in an in vitro transcription reaction performed with purified yeast RNA polymerase II holoenzyme and a classical transcriptional activator. We show that the level of transcription reached by the activator working on low concentrations of holoenzyme can also be reached in the absence of activator by raising the holoenzyme concentration, and that under that condition the activator has no further stimulatory effect. We also show, in agreement with another prediction of the recruitment model, that in a reaction using a holoenzyme purified from cells bearing the "P" mutation, transcription is stimulated by a DNA-tethered peptide that binds the mutant holoenzyme component Gal11P but that lacks a classical activating region.