Synergistic and promoter-selective activation of transcription by recruitment of transcription factors TFIID and TFIIB

Abstract

Eukaryotic transcriptional activators may function by stimulating formation of RNA polymerase II preinitiation complexes at the core promoter of genes. In this case, their mode of action will intrinsically depend on how these complexes assemble on promoters in living cells, an issue that remains largely unexplored. Here we show that in yeast the basal transcription machinery is brought to the promoter in the form of at least two subcomplexes, TFIID and a complex comprising TFIIB and other essential components. Individual recruitment of either complex by artificial contact with a transcriptionally inactive, sequence-specific DNA-binding protein suffices to trigger transcriptional activation from a wild-type core promoter bearing the appropriate binding site. In contrast, activation from a promoter containing a weakened TATA element is only observed upon recruitment of TFIID. Tethering TFIIB on that promoter remains without effect, but the simultaneous recruitment of both components leads to strong synergistic activation. These findings suggest a simple mechanism whereby two activators that contact distinct subcomplexes of the basal machinery may stimulate transcription synergistically and differentially depending on the nature of the promoter.

Figure 1

Recruitment of TFIID to a target promoter by fusing TAF90 or TAF60 to the transcriptionally inactive DNA-binding protein RFX stimulates transcription. The activity of a lacZ reporter gene bearing a single RFX-binding site (X) upstream of the his3 TATA element (14) was assessed in yeast strains expressing the indicated proteins from plasmid DNAs. The proteins were either expressed at low levels from the TBP promoter on a centromeric plasmid, or overproduced by introducing the genes into the multicopy vector pYES2 (Invitrogen) under control of the GAL1 promoter. Transformed yeast cells were grown in selective medium containing galactose and assayed for β-galactosidase activity. In this and the other figures of this paper, values are relative to the level of β-galactosidase activity seen with VP16–RFX under normal conditions: this amount was assigned a value of 100. Tx refers to the remainder of the general factors and pol II.

Figure 2

Transcriptional activation upon recruitment of TFIIB–Myc by Max–RFX. The activity of the RFX-dependent lacZ gene was determined in strains expressing the indicated RFX fusion proteins from the TBP promoter and TFIIB derivatives from the native TFIIB promoter. The parent strain contains a chromosomal TFIIB deletion and expresses low levels of wild-type TFIIB from pYES2 by growing the cells in glucose medium. The ability of the TFIIB–Myc variants to support viability was examined by spotting 10⁴ transformants on plates containing 5-fluoroorotic acid (FOA), a uracil analog that selects against cells carrying pYES2 marked with URA3 and encoding wild-type TFIIB. A protein immunoblot of whole-cell extracts from strains expressing the TFIIB–Myc proteins at higher levels from the ADH1 promoter (lanes 1–8) or TFIIB from its own promoter (lane 9) is shown at the bottom with the positions of TFIIB and TFIIB–Myc indicated. The TFIIB–Myc mutants contain either (lanes 4 and 3) or both (lane 5) F189R, G204R substitutions of residues known from the crystal structure to contact TBP (38), or a single C48S mutation (lane 6) important for interaction with pol II–TFIIF (–42). Nter designates a hybrid protein carrying the Myc dimerization motif at the amino terminus (lane 7).

Figure 4

The nature of the TATA element differentially influences transcriptional activation by recruitment of TFIID or TFIIB. Comparison of transcriptional activation by TAF90 or TFIIB fused covalently to RFX, or by VP16–RFX from wild-type (TATA) and mutant (TGTA) promoters bearing an upstream RFX-binding site (14). The chimeric proteins were expressed at low levels from the TBP promoter on a centromeric plasmid. Because the wild-type proteins interfere with activation mediated by the derivatives fused to RFX (see Figs. 1 and 3), the strains are deleted either for the chromosomal TAF90 gene (lanes 1 and 2), or for the TFIIB gene (lanes 3 and 4), and they express low levels of the corresponding proteins from pYES2 by growing the cells in glucose medium.

Figure 3

TFIIB associates with essential proteins before reaching the promoter. Wild-type and mutant forms of TFIIB expressed at high levels from the ADH1 promoter were examined for their effect on transcription activated by TFIIB–Myc and Max–RFX (lanes 4–6), or by VP16–RFX (lane 8). The TFIIB mutants carry either the C48S substitution that compromises interaction of TFIIB with pol II–TFIIF (–42) (lane 4), or the double amino acid change F189R/G204R on the surface of the protein that contacts TBP (38) (lane 5). The strains containing TFIIB–Myc are deleted for the chromosomal TFIIB gene. The amounts of TFIIB proteins expressed in these cells were determined by the protein immunobloting shown at the bottom.

Figure 5

Simultaneous recruitment of TFIID and TFIIB to a TGTAAA-containing promoter activates transcription synergistically. TFIID is tethered to the promoter by fusing covalently TAF90 to Max–RFX, and TFIIB–Myc is recruited by the same Max–RFX–TAF90 hybrid protein through the Max dimerization domain. TAF90 was also fused at the carboxyl terminus of VP16–RFX to assess activity of VP16 upon artificial recruitment of TFIID. The strains used in these experiments carry a deletion of the TFIIB gene, and they express normal levels of endogenous TAF90 which competes with RFX–TAF90 for its site in the TFIID complex, thus explaining the reduced levels of activation mediated by RFX–TAF90 compared with those observed in Fig. 4.