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

Abstract

Unregulated transcription of protein-encoding genes in vitro is dependent on 12-subunit core RNA polymerase II and five general transcription factors; TATA binding protein (TBP), transcription factor (TF)IIB, TFIIE, TFIIF, and 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 express the corresponding proteins in recombinant form in Escherichia coli and in Sf21 insect cells, and all proteins have been purified to . 90% homogeneity. We have also purified a recombinant His 6 -tagged mouse TBP to near homogeneity and show that it is active in both a reconstituted mouse in vitro transcription system and a TBP-dependent in vitro transcription system from 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 different promoters in vitro. Functional studies of the S-phase-specific mouse ribonucleotide reductase R2 promoter using both the highly purified system described here (a mouse cell nuclear extract in vitro transcription system) and in vivo R2-promoter reporter gene assays together identify an NF-Y interacting promoter proximal CCAAT-box as being essential for high-level expression from the R2 promoter.Keywords: in vitro transcription; RNA polymerase II; ribonucleotide reductase; mouse general transcription factors.RNA polymerase II catalyses transcription of all proteinencoding genes in eukaryotic cells. The polymerase is dependent on accessory proteins, called the general transcription factors (GTFs) for accurate initiation in vitro [1][2][3][4]. One of these factors, TATA binding protein (TBP), is able to bind directly to promoter DNA and to initiate the sequential addition of the remaining components of the transcription initiation complex. The minimal set of GTFs required for basal transcription in vitro includes, in addition to the RNA polymerase and TBP, also TFIIB, TFIIE, TFIIF, and TFIIH. These factors are both necessary and sufficient for proper initiation of transcription in in vitro transcription systems purified from yeast Saccharomyces cerevisiae, rat and human cells [2,[5][6][7][8].The identification of a minimal set of GTFs, cloning of the corresponding cDNAs from different species and the recently described three-dimensional structure of the yeast RNA polymerase II [9] have facilitated studies of the factors involved in forming a functional preinitiation complex. Swapping experiments of GTFs purified from the distantly related yeasts S. cerevisiae and Schizosaccharomyces pombe have shown that none of the GTFs, except TBP, is individually exchangeable between the different systems [10]. However, the combined substitutions of S. pombe TFIIB and RNA polymerase or TFIIE together with TFIIH, with the corresponding S. cerevisiae factors are possible and thus indicate a specie...

Mediator Influences Schizosaccharomyces pombe RNA Polymerase II-dependent Transcription in Vitro

“…Our reactions were less dependent on TFIIH, which had a 3-fold stimulatory effect on basal transcription. In agreement with our observation, TFIIH is not needed for transcription of certain promoters on negatively supercoiled templates in mammalian in vitro transcription systems (27)(28)(29). In contrast, our S. pombe in vitro transcription system appears to be more dependent on TFIIE than the human system (27, 28), since we can observe no transcription in the absence of this factor.…”

S Phase-specific Transcription of the Mouse Ribonucleotide Reductase R2 Gene Requires Both a Proximal Repressive E2F-binding Site and an Upstream Promoter Activating Region

“…Overlap extension PCR was used to introduce a CCAAT-to-CTAGT mutation (16) in pAC10 containing mutated E2F-I and E2F-II sites. The same method was used to obtain pAC10 plasmids containing either a mutated E2F-I or E2F-II site.…”

“…Neither mutation affects the cell cycle-specific expression pattern. In the absence of the TTTAAA box, R2 transcription is directed by sequences downstream of the transcription start site interacting with TAF II s (15,16).…”

“…Mutating the CCAAT box to CTAGT was previously shown to decrease R2 promoter activity to ϳ20% compared with the wild-type activity without changing the S phase-specific expression profile (16). We now combined the mutated CCAAT box with the mutated E2F-I and E2F-II elements in one reporter gene construct and recorded the effects on the promoter strength and cell cycle expression pattern.…”

“…Neither mutation affects the cell cycle-specific expression pattern. In the absence of the TTTAAA box, R2 transcription is directed by sequences downstream of the transcription start site interacting with TAF II s (15,16).Together with a number of other S phase-specific genes such as DNA polymerase-␣ and thymidylate synthase, the R2 gene was activated when quiescent cells were infected with an E2F1- …”