Yeast GAL11 protein is a distinctive type transcription factor that enhances basal transcription in vitro.

Abstract

The yeast auxiliary transcription factor GAL1l, a candidate for the coactivator, was partially purified from yeast cells, and its function was characterized in a cell-free ranscription system. The partially purified GAL1l protein stimulated basal transcription from the CYCI core promoter by a factor of 4-5 at the step of preinitiation complex formation. GALll protein also enhanced transcription activated by general regulatory factor 1, GAL4-AH, or GAL4-VP16 to the same extent as the basal ranscription. Therefore, the apparent potentiation of the activators by GALll was attributable to the stimulation of basal transcription. The wild-type GALll protein (but not a mutant-type protein) produced in bacteria stimulated transcription as effectively as GAL1l from yeast. These results suggest that GALll functions as a positive cofactor of basal and activator-induced transcription in a cell-free transcription system. that potentiates a weak activator GAL4-AH. Surprisingly, the mutation is a missense mutation within GALI and was therefore designated GALIIP (P stands for potentiator). They suggest a direct interaction between GAL1l and GAL4 molecules and suggest that the complex of the two proteins is a strong activator. They further find that GALII is required for normal functioning of PPR1, an activator for the URA3 gene. Puzzlingly, GALI is also involved in the transcriptional repression of genes, since a loss-of-function mutation of GALII, called sptl3, restores the transcription of genes inactivated by insertion of the yeast transposon Ty (22).To get insight into the complex pleiotrophic functions of GALH in vivo, we analyzed the role of GAL1l biochemically. We have partially purified GALli protein and studied how it works in a cell-free transcription system. We have found that GAL1l enhances the basal transcription as well as the transcription activated by GAL4-AH, GAL4-VP16, or GRF1. These results suggest that, since GAL1l regulates the basal transcription, it is not a bona fide coactivator, but rather it belongs to a distinctive class of basal transcription factors. (15,27). The major start site of GALli mRNA was Abbreviations: UAS, upstream activation sequence; GRF1, general regulatory factor 1; GST, glutathione S-transferase. MATERIALS AND METHODSITo whom reprint requests should be addressed. 8382

Identification of a Novel Class of Target Genes and a Novel Type of Binding Sequence of Heat Shock Transcription Factor in Saccharomyces cerevisiae

“…The 5Ј-region of FSH1 from Ϫ350 to ϩ45, in which the SalI and BamHI sites were created at the 5Ј-and 3Ј-ends, respectively, was amplified by PCR from yeast chromosomal DNA. The SalI-BamHI-digested fragment was substituted for the CYC1 promoter region of the CYC1-lacZ reporter (pLG670Z; YEp-URA3-CYC1-lacZ) (28). Point mutations were introduced by the megaprimer PCR method.…”

“…Transformants were grown in enriched synthetic glucose medium lacking uracil and tryptophan at 28 or at 39°C for 90 min. The ␤-galactosidase activity was determined as described previously (28).…”

Two alternative pathways of transcription initiation in the yeast negative regulatory gene GAL80.

“…Yeast nuclear extract was prepared from strain BJ2168 as previously described (45). The transcription reaction was performed for 90 min at 25°C in a 20-,ul reaction mixture as previously described (45) (29,53).…”

“…The culture was supplemented with 2% galactose and further incubated for 4 to 6 h such that cell density reached an OD600 of 1.0. The activity of 3-galactosidase was assayed as previously described (15,45).…”

“…Yeast recombinant TATAbinding protein (TBP) was kindly supplied by H. Handa. GAL4-VP16 (44) was purified as previously described (45 …”