Activator-dependent transcription by mammalian RNA polymerase II: In vitro reconstitution with general transcription factors and cofactors

Abstract

Publisher Summary This chapter describes a simple general system reconstituted with pure recombinant and natural partially purified human factors that reproduces high levels of transcription stimulation by a wide variety of natural and recombinant activators, including Spl, Octl/2 (in the presence of the B-cell-specific OCAB coactivator), NF-κB, Gal4-AH, Gal4-VP16, Ga14-p53, Gal4-CTF1, and Gal4-Spl. This system combines a highly pure TFIID, recombinant TFIIB, and partially purified TFIIA, TFIIE/F/H, and RNA polymerase II; and requires, for high levels of activator-mediated transcription, the presence of either the USA (upstream activator-dependent stimulatory activity) cofactor fraction, which contains both positive (PCs) and negative (NCs) cofactors, or the recombinant human PC4, which is the major coactivator of the USA cofactor fraction. The system described in this chapter should be a useful start point not only for the analysis of the molecular mechanisms involved in transcription activation and the ultimate reconstitution with homogeneous components, but also for the biochemical identification of novel activities essential for the reconstitution of physiological gene regulatory pathways in the context of natural activator/promoter systems and in chromatin-assembled templates. The biochemical identification of novel activities/cofactors with highly purified in vitro systems requires the evaluation of these functions by using and/or developing more physiological assay systems.