Are all DNA binding and transcription regulation by an activator physiologically relevant?

Understanding how a regulatory protein occupies its sites in vivo is centra l to understanding gene regulation. Using the yeast Gal4 protein as a model for such studies, we have found 239 potential Gal4 binding sites in the ye ast genome, 186 of which are in open reading frames (ORFs). This raises the questions of whether these sites are occupied by Gal4 and, if so, to what effect. We have analyzed the Saccharomyces cerevisiae ACC1 gene (encoding a cetyl-coenzyme A carboxylase), which has three Gal4 binding sites in its OR F. The plasmid titration assay has demonstrated that Gal4 occupies these si tes in the context of an active ACC1 gene. We also find that the expression of the ACC1 is reduced fourfold in galactose medium and that this reductio n is dependent on the Gal4 binding sites, suggesting that Gal4 bound to the ORF sites affects transcription of ACC1. Interestingly, removal of the Gal 4 binding sites has no obvious effect on the growth in galactose under labo ratory conditions. In addition, though the sequence of the ACC1 gene is hig hly conserved among yeast species, these Gal4 binding sites are not present in the Kluyveromyces lactis ACC1 gene. We suggest that the occurrence of t hese sites may not be related to galactose regulation and a manifestation o f the "noise" in the occurrence of Gal4 binding sites.