Multiple and Distinct Activation and Repression Sequences Mediate the Regulated Transcription of IME1, a Transcriptional Activator of Meiosis-Specific Genes inSaccharomyces cerevisiae

ABSTRACT IME1 encodes a transcriptional activator required for the transcription of meiosis-specific genes and initiation of meiosis in Saccharomyces cerevisiae. The transcription ofIME1 is repressed in the presence of glucose, and a low basal level of IME1 RNA is observed in vegetative cultures with acetate as the sole carbon source. Upon nitrogen depletion a transient induction in the transcription of IME1 is observed in MATa/MATα diploids but not in MAT-insufficient strains. In this study we demonstrate that the transcription of IME1 is controlled by an extremely unusual large 5′ region, over 2,100 bp long. This area is divided into four different upstream controlling sequences (UCS). UCS2 promotes the transcription of IME1 in the presence of a nonfermentable carbon source. UCS2 is flanked by three negative regions: UCS1, which exhibits URS activity in the presence of nitrogen, and UCS3 and UCS4, which repress the activity of UCS2 in MAT-insufficient cells. UCS2 consists of alternate positive and negative elements: three distinct constitutive URS elements that prevent the function of any upstream activating sequence (UAS) under all growth conditions, a constitutive UAS element that promotes expression under all growth conditions, a UAS element that is active only in vegetative media, and two discrete elements that function as UASs in the presence of acetate. Sequence analysis of IME1 revealed the presence of two almost identical 30- to 32-bp repeats. Surprisingly, one repeat, IREd, exhibits constitutive URS activity, whereas the other repeat, IREu, serves as a carbon-source-regulated UAS element. The RAS-cyclic AMP-dependent protein kinase cAPK pathway prevents the UAS activity of IREu in the presence of glucose as the sole carbon source, while the transcriptional activators Msn2p and Msn4p promote the UAS activity of this repeat in the presence of acetate. We suggest that the use of multiple negative and positive elements is essential to restrict transcription to the appropriate conditions and that the combinatorial effect of the entire region leads to the regulated transcription ofIME1.

Activation mechanism of the multifunctional transcription factor repressor-activator protein 1 (Rap1p).

Transcriptional activation in eukaryotic organisms normally requires combinatorial interactions of multiple transcription factors. In most cases, the precise role played by each transcription factor is not known. The upstream activating sequence (UAS) elements of glycolytic enzyme genes in Saccharomyces cerevisiae are excellent model systems for the study of combinatorial interactions. The yeast protein known as Rap1p acts as both a transcriptional repressor and an activator, depending on sequence context. Rap1p-binding sites are found adjacent to Gcr1p-binding sites in the UAS elements of glycolytic enzyme genes. These UAS elements constitute some of the strongest activating sequences known in S. cerevisiae. In this study, we have investigated the relationship between Rap1p- and Gcr1p-binding sites and the proteins that bind them. In vivo DNA-binding studies with rap1ts mutant strains demonstrated that the inability of Rap1p to bind at its site resulted in the inability of Gcr1p to bind at adjacent binding sites. Synthetic oligonucleotides, modeled on the UAS element of PYK1, in which the relative positions of the Rap1p- and Gcr1p-binding sites were varied prepared and tested for their ability to function as UAS elements. The ability of the oligonucleotides to function as UAS elements was dependent not only on the presence of both binding sites but also on the relative distance between the binding sites. In vivo DNA-binding studies showed that the ability of Rap1p bind its site was independent of Gcr1p but that the ability of Gcr1p to bind its site was dependent on the presence of an appropriately spaced and bound Rap1p-binding site. In vitro binding studies showed Rap1p-enhanced binding of Gcr1p on oligonucleotides modeled after the native PYK1 UAS element but not when the Rap1p- and Gcr1p-binding sites were displaced by 5 nucleotides. This work demonstrates that the role of the Rap1p in the activation of glycolytic enzyme genes is to bind in their UAS elements and to facilitate the binding of Gcr1p at adjacent binding sites.