Identification of sequence elements that confer cell-type-specific control of MF alpha 1 expression in Saccharomyces cerevisiae.

Abstract

The MFal gene of Saccharomyces cerevisiae, a major structural gene for mating pheromone a factor, is an a-specific gene whose expression is regulated by the mating-type locus. To study the role of sequences upstream of MFal in its expression and regulation, we generated two sets of promoter deletions: upstream deletions and internal deletions. By analyzing these deletions, we have identified a TATA box and two closely related, tandemly arranged upstream activation sites as necessary elements for MFaJ expression. Two upstream activation sites were located ca. 300 and 250 base pairs upstream of the MFal transcription start points, which were also determined in this study. Each site contained a homologous 22-base-pair sequence, and both sites were required for maximum transcription level. The distance between the upstream activation sites and the transcription start points could be altered without causing loss of transcription efficiency, and the sites were active in either orientation with respect to the coding region. These elements conferred cell type-specific expression on a heterologous promoter. Analysis with host mating-type locus mutants indicates that these sequences are the sites through which the MATal product exerts its action to activate the MFal gene. Homologous sequences with these elements were found in other a-specific genes, MFa2 and STE3, and may mediate activation of this set of genes by MATaJ.

Regulation of alpha-factor production in Saccharomyces cerevisiae: a-factor pheromone-induced expression of the MF alpha 1 and STE13 genes.

“…2, lane 3). As reported earlier (2,22,24,31) (20,24). Here, this fragment was joined via a multiple cloning site present in plasmid pSEY304 (4) to a DNA fragment containing almost the entire sequence for the mature form of invertase.…”

“…Basal activity of aspecific genes like MFal requires the concerted binding of the MATal gene product and a protein factor termed PRTF to specific P-and Q-box sequences in addition to the action of the STE4, STE5, STE7, STEJJ, and STE12 gene products (7). In MATa/MATTa diploids, MFal might be repressed by the complex of the MATal and the MATa2 gene products since a DNA sequence with significant homology to the recognition sequence of this repressor complex can be found in the MFal promoter (31).…”

“…Support for this argument also comes from the observation that, upon removal of a 94-base-pair DNA segment of the CHSJ promoter containing four repeats of the TGAAACA motif, pheromone-induced expression of a CHSI::SUC2 fusion gene is completely abolished (3). Six repeats of the consensus heptamer are located in the upstream region of MFal] (at positions -211, -251, -638, -656, -707, -910, where the numbers mark the position of the last A relative to the ATG [31]). By analogy, it can be assumed that they might serve as cis-acting elements involved in a-factor-induced MFal expression.…”

A protein involved in minichromosome maintenance in yeast binds a transcriptional enhancer conserved in eukaryotes.

“…Each footprinted region contains a sequence motif related to the 10-bp dyad symmetry element CCTAATTAGG. Two of these regions correspond to MF~I UAS2 (proximal UAS) and UAS1 (distal UAS), the two MF~I UASs that require ~ 1 protein for activity, and are silent in diploid cells (Inokuchi et al 1987).…”

The yeast transcription activator PRTF, a homolog of the mammalian serum response factor, is encoded by the MCM1 gene.

“…Insight as to how ~1 activates transcription of a-specific genes has come from an analysis both of the upstream activation sequences (UAS elements) of a-specific genes (UAS; Inokuchi et al 1987;Jarvis et al 1988;Flessel et al 1989) and of the proteins that bind to those UAS elements (Bender and Sprague 1987;Tan et al 1988). These studies revealed that al binds at a-specific 1present address: Solar Energy Research Institute, Golden, Colorado 80401 USA.…”