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 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 His6-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.
Kotova
I
I
Department of Medical Biosciences, Medical Biochemistry, Umeå University, Sweden.
Chabes
A L
AL
Segerman
B
B
Flodell
S
S
Thelander
L
L
Björklund
S
S
D000818:Animals; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000502:physiology*; D051379:Mice; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification; D012264:Ribonucleotide Reductases / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D035581:Transcription Factor TFIIB; D035362:Transcription Factor TFIID; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000502:physiology*; D019473:Transcription Factors, TFII; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C490039:TAF6 protein, S cerevisiae; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D035581:Transcription Factor TFIIB; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII; C066644:transcription factor TFIIE; D051758:Transcription Factor TFIIH; D012264:Ribonucleotide Reductases; C108427:ribonucleotide reductase R2 subunit; D012319:RNA Polymerase II; C056604:transcription factor TFIIF