An artificial transcriptional activating region with unusual properties

Abstract

We describe a series of transcriptional activators generated by adding amino acids (eight in one case, six in another) to fragments of the yeast Saccharomyces cerevisiae activator Gal4 that dimerize and bind DNA. One of the novel activating regions identified by this procedure is unusual, compared with previously characterized yeast activating regions, in the following ways: it works more strongly than does Gal4's natural activating region as assayed in yeast; it is devoid of acidic residues; and several lines of evidence suggest that it sees targets in the yeast transcriptional machinery at least partially distinct from those seen by Gal4's activating region.

Figure 1

Isolation of Gal4-fused peptide activators. (A) A lacZ reporter bearing five consensus 17-bp Gal4 binding sites 191 bp upstream of the GAL1 TATA box. (B) Sequences of activating regions and their transcriptional activities in yeast as assayed by using the reporter in A. (C) Amino acid compositions of the peptides.

Figure 2

Activities of mutants of Gal4(1–100)+P201. (A) Scrambled P201. (B) Arginine substitutions. (C) Carboxyl-terminal truncations. (D) Single internal deletions in the region of Gal4(96–100). (E) Alanine substitutions.

Figure 3

A hypothetical helical structure of GAL4(1–100)+P201. (A) A putative helical wheel formed by GAL4(1–100)+P201. (B) Effects of proline substitution at Q99 and D100.

Figure 4

Activity of a fragment of Gal4(1–100)+P201 inserted in place of the natural activating region of the yeast activator Pho4. Cells were grown and assayed for acid phosphatase assay as described by Svaren et al. (37).

Figure 5

Activation by Gal4-peptides in vitro using purified yeast holoenzyme. The holoenzyme (1 μg) was supplemented with TBP (1 pmol), TFIIE (0.5 pmol), and DNA template shown above (100 ng) in the absence or presence of Gal4-VP16 (50 ng), Gal4(1–100)+P201 (20 ng), or Gal4(1–100)+(840–850)+P64 (20 ng). Arrow indicates the E4 extension product.

Figure 6

Interaction with targets in the transcriptional machinery. (A) The effect of overexpression of one activator on the activity of another. The overexpressed activator is called the squelcher, and the reporter is as indicated. (B) Interaction of Gal4-peptides with TBP, SRB4, and SRB10. Labeled Gal4(1–100), Gal4(1–100)+P201, or Gal4(1–100)+(840–881) were incubated with flag-tagged targets of Gal4 and then tested for their ability to coimmunoprecipitate with the targets (15). Input lane contains 10% of the TnT material used in each coimmunoprecipitation.