Transcriptional activation by artificial recruitment in yeast is influenced by promoter architecture and downstream sequences

Abstract

The idea that recruitment of the transcriptional machinery to a promoter suffices for gene activation is based partly on the results of "artificial recruitment" experiments performed in vivo. Artificial recruitment can be effected by a "nonclassical" activator comprising a DNA-binding domain fused to a component of the transcriptional machinery. Here we show that activation by artificial recruitment in yeast can be sensitive to any of three factors: position of the activator-binding elements, sequence of the promoter, and coding sequences downstream of the promoter. In contrast, classical activators worked efficiently at all promoters tested. In all cases the "artificial recruitment" fusions synergized well with classical activators. A classical activator evidently differs from a nonclassical activator in that the former can touch multiple sites on the transcriptional machinery, and we propose that that difference accounts for the broader spectrum of activity of the typical classical activator. A similar conclusion is reached from studies in mammalian cells in the accompanying paper [Nevado, J., Gaudreau, L., Adam, M. & Ptashne, M. (1999) Proc. Natl. Acad. Sci. USA 96, 2674-2677].

Figure 1

Gene activation by various nonclassical activators in yeast. (A) Transcriptional activation elicited by Pho4 derivatives at the PHO5 gene. All the Pho4 fusions were expressed from the PHO4 promoter on a high-copy (2 μ) vector (24). Activity was measured as acid phosphatase units produced by the product of the PHO5 gene directly off its chromosomal locus under high phosphate conditions in a pho80 strain (YAG23). Wild-type Pho4 was carried on an ARS-CEN plasmid because overexpression of Pho4 causes severe growth defects (24). (B) Transcriptional activation elicited by LexA derivatives at an HIS3-based reporter template. All the LexA fusions were expressed from the ADH1 promoter on a high-copy (2 μ) vector. Transcriptional activity was measured as β-galactosidase units produced by the lacZ gene product in yeast strain FT4a. The reporter construct was carried on a 2 μ plasmid.

Figure 2

Position of the activator-binding sites influences transcriptional activation elicited by nonclassical activators. All the Gal4 constructs (except Gal4 + Gal11) were expressed from the ADH1 promoter on a high-copy (2 μ) vector. Gal4 + Gal11 was expressed from a β-actin promoter and carried on an ARS-CEN plasmid (15). Transcriptional activity was measured as β-galactosidase units produced off chromosomally integrated templates in yeast strains YAG101 (template A; white bars) and YAG4 (template B; hatched bars).

Figure 3

The promoter sequence influences transcriptional activation elicited by nonclassical activators. (A) Transcriptional activation elicited by LexA derivatives elicited at chromosomally integrated (at the ura3–52 locus) reporter templates bearing either the GAL1 or PHO5 promoters. Both templates bear two LexA binding sites upstream of the respective TATA element, but on template A the DNA separating these sites from the ATG is taken from the GAL1 gene, and the corresponding sequence on template B is from the PHO5 gene. The distances between the LexA sites and the TATA elements are those found naturally at the GAL1 gene (191 bp; template A) and the PHO5 gene (133 bp; template B). All the LexA constructs were expressed from the ADH1 promoter on a 2 μ vector. Transcriptional activity was measured as β-galactosidase units in yeast strains YAG22 (template A; white bars) and YAG35 (template B; black bars). (B) Primer extension analysis. The cDNA-labeled lacZ denotes transcripts from the GAL1∷lacZ reporter of YAG22.

Figure 4

Coding sequences downstream of the ATG influence transcriptional activation elicited by nonclassical activators. All the Pho4 fusions were expressed from the PHO4 promoter on a high-copy (2 μ) vector and wild-type Pho4 was carried on an ARS-CEN plasmid (24).

Figure 5

Synergistic activation of nonclassical activators with a DNA-tethered activating region. All the Gal4 and LexA constructs were expressed from the ADH1 promoter on a high-copy (2 μ) vector. Transcriptional activity was measured as β-galactosidase units produced off a chromosomally integrated template in yeast strain ZZY41.

Figure 6

Overexpression of non-DNA-tethered activating regions does not enhance transcriptional activation elicited by nonclassical activators. The Gal4 constructs were expressed from the ADH1 promoter on a high-copy (2 μ) vector; Gal4 + Gal80 was carried on an ARS-CEN vector. RII + RII is comprised of two tandemly fused copies of the Gal4-activating region and RII + Gcn4 comprises the same RII moiety fused to Gcn4 lacking its DNA-binding domain. Both of these activating regions were expressed from the β-actin promoter and carried on a 2 μ plasmid. Transcriptional activity was measured as β-galactosidase units in yeast strain ZZY41.