Abstract: Gene expression in eukaryotes is normally believed to be controlled by transcriptional regulators that activate genes encoding structural proteins and enzymes. To identify previously unrecognized DNA binding activities, a yeast proteome microarray was screened with DNA probes; Arg5,6, a well-characterized mitochondrial enzyme involved in arginine biosynthesis, was identified. Chromatin immunoprecipitation experiments revealed that Arg5,6 is associated with specific nuclear and mitochondrial loci in vivo, and Arg5,6 binds to specific fragments in vitro. Deletion of Arg5,6 causes altered transcript levels of both nuclear and mitochondrial target genes. These results indicate that metabolic enzymes can directly regulate eukaryotic gene expression.