Identification of trans-acting factors involved in HEM13 expression

HEM13 encodes coproporphyrinogen oxidase, the sixth enzyme of the heme biosynthetic pathway. Expression of HEM13 is repressed under aerobic conditions. In the absence of heme or oxygen, the expression of HEM13 increases forty-fold. Mutations defining trans-acting factors involved in the regulation of HEM13 expression were isolated and analyzed. This analysis identified two different alleles of HAP1. HAP1 encodes an activator protein involved in the expression of a number of heme-dependent genes such as CYC1, which encodes iso-1-cytochrome c, and ROX1, which encodes a repressor of HEM13 expression. The hap1 mutant allele in one strain confers a phenotype much like that of the hap1::LEU2 allele in that HEM13 expression is elevated under repressing conditions and not fully inducible in the absence of heme. In addition, expression of ROX1 is constitutively low. The other mutant allele of HAP1 confers a novel phenotype. Expression of HEM13 is uninducible in a strain containing this other allele of HAP1 while that of ROX1 and CYC1 has become heme-independent. The mutation associated with this mutant allele of hap1 was localized to a glycine to aspartate change in amino acid 235 of HAP1. DNA binding assays indicated that the protein made from this HAP1 allele retains the ability to bind DNA, but that unlike wild-type HAP1 protein, this binding is not stimulated by heme. The regulatory sequences involved in HEM13 expression have been localized to a 365 bp region which contains several distinct positive and negative regulatory elements. Gel shift experiments with overlapping fragments containing sequences from this region of the HEM13 promoter indicate that multiple factors interact with the elements in this region