SYNERGISTIC ACTIVATION OF ADH2 EXPRESSION IS SENSITIVE TO UPSTREAM ACTIVATION SEQUENCE-2 (UAS2) ORIENTATION, COPY NUMBER, AND UAS1-UAS2 HELICAL PHASING

The alcohol dehydrogenase 2 (ADH2) gene of Saccharomyces cerevisiae is under stringent glucose repression, Two cis-acting upstream activatio n sequences (UAS) that function synergistically in the derepression of ADH2 gene expression have been identified. UAS1 is the binding site f or the transcriptional regulator Adr1p. UAS2 has been shown to be impo rtant for ADH2 expression and confers glucose-regulated, ADR1-independ ent activity to a heterologous reporter gene. An analysis of point mut ations within UAS2, in the context of the entire ADH2 upstream regulat ory region, showed that the specific sequence of UAS2 is important for efficient derepression of ADH2, as would be expected if UAS2 were the binding site for a transcriptional regulatory protein, In the context of the ADH2 upstream regulatory region, including UAS1, working in co ncert with the ADH2 basal promoter elements, UAS2-dependent gene activ ation was dependent on orientation, copy number, and helix phase, Mult imerization of UAS2, or its presence in reversed orientation, resulted in a decrease in ADH2 expression. In contrast, UAS2-dependent express ion of a reporter gene containing the ADH2 basal promoter and coding s equence was enhanced by multimerization of UAS2 and was independent of UAS2 orientation. The reduced expression caused by multimerization of UAS2 in the native promoter was observed only in the presence of ADR1 . Inhibition of UAS2 dependent gene expression by Adr1p was also obser ved with a UAS2-dependent ADH2 reporter gene. This inhibition increase d with ADR1 copy number and required the DNA-binding activity of Adr1p . Specific but low-affinity binding of Adr1p to UAS2 in vitro was demo nstrated, suggesting that the inhibition of UAS2-dependent gene expres sion observed in vivo could be a direct effect due to Adr1p binding to UAS2.