Cloning of the gene encoding the yeast protein BTF1Y, which can substitute for the human TATA box-binding factor.

Abstract

An activity (designated BTF1Y) in extracts of Saccharomyces cerevisiae can substitute for the human TATA box-binding factor BTF1 in a reconstituted transcription system containing the adenovirus 2 major late promoter, RNA polymerase B (II), and the basic transcription factors BTF2, BTF3, and STF. We have purified BTF1Y to homogeneity, using as assays reconstitution of in vitro transcription and DNase I footprinting on the TATA element. Both activities copurified with a 27-kDa polypeptide as determined by SDS/PAGE. Gel Accurate and specific initiation of transcription in vitro from minimal promoter elements of protein-coding genes requires at least four transcription factors in addition to RNA polymerase B (II) (1-5). The first step in transcription initiation corresponds to the binding of the factor BTF1 (the TATA box-binding factor, also known as TFIID and DB) to the TATA box element (6-8). This binding step is crucial in the initiation pathway, since it results in the formation of a stable "committed" complex onto which the other factors and RNA polymerase B assemble to form a multiprotein complex at the promoter proximal site (3, 4, 9-11).Although numerous attempts have been made to purify mammalian BTF1, notably from human HeLa cell extracts, purification was only partial (e.g., ref. MATERIALS AND METHODSPurification of BTF1Y Protein. S100 extract (34 mg of protein per ml) was prepared from frozen S. cerevisiae cells as described (19). After overnight dialysis against buffer DB [10 mM Tris'HCl, pH 7.9/5 mM MgCl2/0.5 mM dithiothreitol/17.4% (vol/vol) glycerol/0.2 mM phenylmethanesulfonyl fluoride (PMSF)] containing 0.05 M KCI, 125 ml of 2-folddiluted S100 was loaded onto a 450-tnl heparin-Ultrogel (IBF) column equilibrated in buffer DB containing 0.1 M KCI. After washing, proteins were eluted stepwise with buffer DB containing 0.15 M KCI, 0.37 M KCI (HEPO.37 fraction), and 0.6 M KCl. BTF1Y activity was found in the HEPO.37 fraction. After dialysis in buffer B (50 mM Tris-HCl, pH 7.9/8.7% glycerol/0.1 mM EDTA/0.5 mM dithiothreitol/0.1 mM PMSF) containing 0.08 M KCI, 2 liters of HEPO.37 fraction was loaded onto a 500-mI DEAE-Sephacel column. The flow-through fraction (DEFT80), which contained the BTF1Y activity, was made 50 mM potassium phosphate (pH 6.3) and loaded onto a sulfopropyl-5PW (Toyo Soda, Tokyo) HPLC column equilibrated in the same buffer. Proteins were eluted with a linear 0.08-0.60 M KCl gradient. Active BTF1Y fractions, which eluted at 0.4-0.5 M KCl (SPO.4 fraction), were dialyzed against buffer B (without PMSF) containing 0.05 M KCl, and 25 ml was loaded onto a heparin-5PW (Toyo Soda) HPLC column. Proteins were eluted with a linear 0.05-0.60 M KCI gradient, with BTF1Y activity recovered at -0.5 M KCl (HEPO.5 fraction). Ten milliliters of the HEPO.5 fraction was dialyzed against buffer C (50 mM Tris-HCl, pH 7.9/17.4% glycerol/0.1 mM EDTA/0.5 mM dithiothreitol/5 mM MgCI2) containing 0..1 M KCl and loaded onto a 400 /ul/heparin-Ultrogel column, equilibrated in the same buffer. Proteins w...

Multimerization of the mouse TATA-binding protein (TBP) driven by its C-terminal conserved domain

“…We found that the C-terminus of TBP was essential for TBP-TBP interaction. The C-terminal domain of TBP has multiple motifs/domains for DNA binding (basic repeat, direct repeat and sigma homology) (38)(39)(40). X-ray crystallography has shown that TBP forms a saddle shape structure (23).…”

Basic transcription factor 3 (BTF3) regulates transcription of tumor-associated genes in pancreatic cancer cells

“…13 We have previously identified BTF3 as a differentially expressed gene in PDAC and chronic pancreatitis (CP). 14,15 In the present study we: (1) analyzed the expression of BTF3 mRNA and protein in the normal and diseased pancreas (CP and PDAC) and pancreatic cancer cell lines, and the localization of BTF3 in these tissues; (2) analyzed the functional consequences of BTF3 silencing in pancreatic cancer cell lines in terms of resistance to chemo/radiotherapy-induced apoptosis, and (3) used a DNA micro-array analysis to identify regulation of genes after BTF3 silencing, in order to specify the role of BTF3 in the transcriptional regulation of tumor-associated genes.…”

“…Basic transcription factor 3 (BTF3), also known as nascent polypeptide-associated complex b (NACB), is a 27 KD protein 1,2 that is involved in the initiation of transcription by RNA polymerase from proximal promoter elements such as the TATA box and CAAT box sequences. 1,3,4 BTF3 is present in two isoforms: BTF3a, which has the transcription characteristics of BTF3, and BTF3b, which lacks the first 44 amino acids of BTF3a and is transcriptionally inactive.…”

Early transcription factor subunits are encoded by vaccinia virus late genes.

“…There are no significant homologs of either subunit of VETF in a library of translated GenBank version 63.0 sequences, which includes the amino acid sequences of a number ofbacterial a factors. In addition, no significant homology was found between VETF and the sequenced eukaryotic general transcription initiation factors, human RAP30 (40) and yeast TFIID (41)(42)(43)(44).…”