A 22-base-pair (bp) inverted repeat present in the ADH2 promoter is an upstream activation sequence (UAS1) which confers ADRl-dependent activation upon a heterologous Saccharomyces cerevisiae promoter. UAS1 was nonfunctional when placed within an intron 3' to the transcription start site. The ll-bp sequence which constitutes one-half of the UAS1 palindrome did not activate transcription in a single copy, as direct repeats, or in an inverted orientation opposite to that of ADH2 UAS1. Furthermore, two pairs of symmetrical point mutations within UAS1 significantly reduced activation. This result suggests that a specific orientation of sequences within UAS1 is necessary for ADRl-dependent activation. We determined that an ADRl-dependent complex was formed with UAS1 and, to a lesser extent, with the nonfunctional ll-bp half palindrome. However, the 11 bp did not confer UAS activity, suggesting that ADRI binding is not sufficient for activation in vivo. ADR1 did not bind to mutant UAS1 sequences in vitro, indicating that their decreased activation is attributable to a reduced affinity of ADR1 for these sequences. We also identified an additional 20-bp ADH2 element (UAS2) that increased the expression of CYCl-lacZ 20-fold when combined with UAS1. UAS2 permitted ADRl-independent, glucose-regulated expression of the hybrid gene. Consistent with this observation, ADR1 did not form a detectable complex with UAS2. Deletion of UAS2 at the chromosomal ADH2 locus virtually abolished ADH2 derepression and had no effect on glucose repression.Recent studies indicate that eucaryotic gene regulation often involves the coordinate interaction of multiple transcription factors (4,7,34,(36)(37)(38)(39). These trans-acting proteins may regulate several genes which function in separate pathways (4, 7). Developmental or metabolic regulation of a gene may be determined by a combination of interactions between transcription factors due to the arrangement of their binding sites within a promoter. These factors act by binding to regulatory sequences which often occur as multiple copies of short (5-to 20-base-pair [bp]) nucleotide sequences present as direct or indirect repeats (3,16,17,23,35,52). In Saccharomyces cerevisiae, DNA sequences that are necessary for transcription are called upstream activation sequences (UAS) (22). Yeast UAS have many of the same properties as enhancers in higher eucaryotes. They function in either orientation and at variable distances with respect to the TATA box and transcription start site (22, 24,43,46); however, UAS have not been found to be functional when placed 3' to the transcription start site.ADH2 is a catabolically regulated gene which encodes the alcohol dehydrogenase II enzyme (ADHII) in S. cerevisiae (12, 31). ADH2 mRNA synthesis is repressed in cells which utilize glucose as their primary carbon source and is derepressed at least 200-fold when glucose is absent (15). Genetic analysis of ADH2 regulation has identified several genes that encode trans-acting factors which are required for full derepre...