GAL4-VP16 stimulates two RNA polymerase II promoters primarily at the preinitiation complex assembly step

We previously demonstrated that RNA polymerase II promoters may be limited in strength not only at the step of transcription complex assembly, but also at initiation or promoter clearance. Here we report on experiments designed to test the possibility that steps following transcription complex assembly might be stimulated by transcriptional activators. Using an in vitro system in which we can independently measure the efficiency of assembly, initiation, and promoter clearance, we have investigated the mechanism by which the model activator GAL4-VP16 increases transcription from two promoters: a weak variant of Ad 2 ML with an altered TATA box, which is inefficient in transcription initiation, and the mouse beta-globin promoter, which is inefficient in promoter clearance. We found that whereas GAL4-VP16 is effective in stimulating both promoters, this increase resulted only from greater transcription complex assembly; the initiation and clearance steps were not affected. Because recent studies have suggested that the core transcription factors TFIIE and TFIIH might be important in promoter clearance, we also attempted to increase the initiation and clearance efficiencies of the Ad ML-TATA mutant and globin promoters by direct addition of excess TFIIE and TFIIH to partially purified preinitiation complexes assembled at each of these promoters. These factors had no effect on transcription by either of the preinitiation complexes.