Lack of a requirement for strict rotational alignment among transcription factor binding sites in yeast

The MF alpha 1 gene of Saccharomyces cerevisiae is an alpha specific gene whose expression is regulated by two upstream activation sites (UASMF alpha 1S), which are the binding sites for activator proteins, MAT alpha 1 and MCM1. UASMF alpha 1 elements act in a synergistic fashion; lack of either element causes reduced expression levels that are 6- to 45-fold less than that of intact promoter in alpha cells. We investigated the effect of manipulating rotational alignments among transcription factor binding sites of MF alpha 1 on the expression of the gene. The expression level of MF alpha 1 decreased with the spacing between the two UASMF alpha 1S and no synergism was observed when the distance of the two elements was longer than 90 base pairs. No strict rotational alignment was required for their synergistic action. We also show that the spacing between UASMF alpha 1 elements and TFIID binding site, the TATA box, had little, if any, effect on MF alpha 1 expression. We chose a sufficient number of positions for UASMF alpha 1S to ensure that, in several of these positions, MAT alpha 1 and MCM1 were on the opposite side of the DNA helix with respect to the TATA box.