A mouse in vitro transcription system reconstituted from highly purified RNA polymerase II, TFIIH and recombinant TBP, TFIIB, TFIIE and TFIIF

Unregulated transcription of protein-encoding genes in vitro is dependent o n 12-subunit core RNA polymerase II and five general transcription factors; TATA binding protein (TBP), transcription factor (TF)IIB, TFIIE, TFIIF, an d TFIIH. Here we describe cloning of the mouse cDNAs encoding TFIIB and the small and large TFIIE and TFIIF subunits. The cDNAs have been used to expr ess the corresponding proteins in recombinant form in Escherichia coli and in Sf21 insect cells, and all proteins have been purified to > 90% homogene ity. We have also purified a recombinant His(6)-tagged mouse TBP to near ho mogeneity and show that it is active in both a reconstituted mouse in vitro transcription system and a TBP-dependent in vitro transcription system fro m Saccharomyces cerevisiae. The more complex general transcription factors, TFIIH and RNA polymerase II, were purified more than 1000-fold and to near homogeneity, respectively, from tissue cultured mouse cells. When combined , the purified factors were sufficient to initiate transcription from diffe rent promoters in vitro. Functional studies of the S-phase-specific mouse r ibonucleotide reductase R2 promoter using both the highly purified system d escribed here (a mouse cell nuclear extract in vitro transcription system) and in vivo R2-promoter reporter gene assays together identify an NF-Y inte racting promoter proximal CCAAT-box as being essential for high-level expre ssion from the R2 promoter.