We have examined the relative contributions of MCM1 and STE12 to the transcription of the a-specific STE2 gene by using a 367 bp fragment from the STE2 5'-noncoding region to drive expression of a reporter lacZ gene. Mutation of the MCM1 binding site destroyed MCM1.alpha 2-mediated repression in alpha cells and dramatically reduced expression in a cells. The residual expression was highly stimulated by exposure of cells to pheromone. Likewise, the loss of STE12 function reduced lacZ expression driven by the wild-type STE2 fragment. In the absence of both MCM1 and STE12 functions, no residual expression was observed. Thus, the STE2 fragment appears to contain two distinct upstream activation sequences (UASs), one that is responsible for the majority of expression in cells not stimulated by pheromone, and one that is responsible for increased expression upon pheromone stimulation. In further support of this idea, a chemically synthesized version of the STE2 MCM1 binding site had UAS activity, but the activity was neither stimulated by pheromone nor reduced in ste12 mutants. Although transcription of alpha-specific genes also requires both MCM1 and STE12, these genes differ from a-specific genes in that they have a single, MCM1-dependent UAS system. The activity of the minimal 26 bp UAS from the alpha-specific STE3 gene was both stimulated by pheromone and reduced in ste12 mutants. These data suggest that at alpha-specific genes STE12 and MCM1 exert their effects through a single UAS.

MCM1 performs several functions necessary for its role in regulating cell type-specific gene expression in the yeast Saccharomyces cerevisiae: DNA binding, transcription activation, and interaction with coregulatory proteins such as al. We analyzed a set of MCM1 deletion derivatives using in vivo reporter gene assays and in vitro DNA-binding studies to determine which regions of MCM1 are important for its various activities. We also analyzed a set of LexA-MCM1 hybrids to examine the ability of different segments of MCM1 to activate transcription independent of MCM1's DNA-binding function. The molecular mechanisms that underlie this transcription regulation hinge on the nature and sequence context of MCM1 binding sites within the upstream control regions of a-and a-specific genes. In the case of a-specific genes, the control regions contain nearly perfect versions of the palindromic MCM1 binding site, the P box. MCM1 binds to these P boxes in vitro (1, 2,15,16,36), and they are crucial for transcription of the gene set in a cells (12,17). The MCM1 binding sites are also essential for repression of a-specific genes in a cells. The P boxes of a-specific genes are flanked by binding sites for a2 protein. MCM1 and a2 bind cooper-*

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MCM1 performs several functions necessary for its role in regulating cell type-specific gene expression in the yeast Saccharomyces cerevisiae: DNA binding, transcription activation, and interaction with coregulatory proteins such as alpha 1. We analyzed a set of MCM1 deletion derivatives using in vivo reporter gene assays and in vitro DNA-binding studies to determine which regions of MCM1 are important for its various activities. We also analyzed a set of LexA-MCM1 hybrids to examine the ability of different segments of MCM1 to activate transcription independent of MCM1's DNA-binding function. The first third of MCM1 [MCM1(1-96)], which includes an 80-residue segment homologous to the mammalian serum response factor, was sufficient for high-affinity DNA binding, for activation of reporter gene expression, and for interaction with alpha 1 in vitro and in vivo. However, the ability of MCM1(1-96) to activate transcription and to interact with alpha 1 was somewhat reduced compared with wild-type MCM1 [MCM1(1-286)]. Optimal interaction with alpha 1 required residues 99 to 117, in which 18 of 19 amino acids are acidic in character. Optimal transcription activation required a segment from residues 188 to 286, in which 50% of the amino acids are glutamine. Deletion of this segment from MCM1 reduced expression of reporter genes by about twofold. Moreover, LexA-MCM1 hybrids containing this segment were able to activate expression of reporter genes that rely on LexA binding sites as potential upstream activation sequences. Thus, glutamine-rich regions may contribute to the activation function of yeast transcription activators, as has been suggested for glutamine-rich mammalian proteins such as Sp1.

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