FIG. 3.

The K151L,K156Y mutant is functional for activated transcription at the permissive temperature. wt, wild type. (A) Strains were spotted on plates containing the media indicated at the density shown, followed by incubation at 30°C. Yeast extract-peptone-dextrose (YPD) is used as a growth rate indicator on rich media. Growth on 15 mM AT, galactose, and CuSO4 requires functional interactions with the Gcn4, Gal4, and Ace1 transcription factors, respectively. (B) Analysis of Gcn4-dependent activation of HIS3 transcription. Constitutive HIS3 expression initiates equally from both the +1 and +13 start sites, whereas Gcn4-activated transcription is mediated primarily through the +13 initiation site. Strains were grown to log phase in synthetic complete medium in either the presence (+) or absence (−) of 15 mM AT. Total RNA (30 μg) was hybridized with 100-fold excesses of HIS3 and DED1 probes and then subjected to S1 nuclease digestion. The DED1 transcript is not affected by AT and is used as a loading control in this experiment.