The CYC7 gene of Saccharomyces cerevisiae encodes the minor species, iso-2, of the cytochrome c protein. Its expression is governed by two regulatory sequences upstream from the gene: a positive site which stimulates transcription 240 base pairs 5' from the protein-coding sequence (-240) and a negative site which inhibits transcription at -300. In this study, the nature of the positive site and its relationship to the negative site has been investigated. Expression of the CYC7 gene is weakly inducible by oxygen. This effect was greatly enhanced by the semidominant CYPI-16 mutation in the trans-acting gene CYPI. The weak oxygen regulation in wild-type cells and the enhanced induction in CYPI-16 mutants were found to be mediated through the positive site. A mutational analysis of this site implicated at least part of a tandem, direct repeat of 9 base pairs as essential for the functioning of this site. The relationship between the positive and negative sites was investigated by comparing the expression of the intact gene with that of derivatives lacking either one or the other site. The expression of the gene containing only the negative site was actually stimulated anaerobically, while the gene containing the positive site alone, although having higher expression aerobically than anaerobically, had higher anaerobic expression than did the intact gene. Thus, it appeared that the combination of the positive and negative sites suppressed anaerobic expression. A model which attempts to explain these properties of the two sites and account for the regulation of the expression of the intact gene is presented.Saccharomyces cerevisiae genes that encode proteins appear to require an upstream activation element for their expression (see reference 10 for a review). Usually located within the few hundred base pairs upstream from the TATA box, which sets the site for the start of transcription (28,31), these elements can serve to stimulate transcription of their gene in either a constitutive or regulated fashion. For a few regulated genes, a protein has been identified which binds to the upstream element (8,17), and in a larger number of cases, a combination of genetic and biochemical evidence suggests that this is the case (for examples, see references 5, 10, 11, 22, 24, 25, and 36). Some genes have, in addition to the activation site, a site at which repression occurs (19, 46), and in the case of the STE6 gene, the MATa2 gene product has been shown to bind to this site (19). Thus, both positive and negative control of transcription can occur.S. cerevisiae contains two nuclear genes encoding the cytochrome c protein, CYCJ encoding iso-1 and CYC7 encoding iso-2 cytochrome c (6, 41). These two genes are expressed at very different levels in respiring cells; iso-1 composes 95% of the cellular cytochrome c (39). This difference is attributable to the much higher rate of transcription of the CYCI gene (30, 50), which in turn is a function of the upstream regulatory elements of the two genes. CYCI contains two activation sites ...