CHARACTERIZATION OF AN UPSTREAM ACTIVATION SEQUENCE AND 2 ROX1P-RESPONSIVE SITES CONTROLLING THE INDUCTION OF THE YEAST HEM13 GENE BY OXYGEN AND HEME DEFICIENCY

The Saccharomyces cerevisiae HEM13 gene codes for coproporphyrinogen o xidase, an oxygen-requiring enzyme catalyzing the sixth step of heme b iosynthesis, Its transcription has been shown to be induced 40-50-fold in response to oxygen or heme deficiency, in part through relief of r epression exerted by Rox1p and in part by activation mediated by an up stream activation sequence (UAS), This report describes an analysis of HEM13 UAS and of the Rox1p-responsive sites by electrophoretic mobili ty shift assays, DNase I footprinting, and mutational mapping, HEM13 U AS is composed of two subelements: a 16-base pair sequence binding a c onstitutive factor acting as a transcriptional activator, and a 5'-fla nking 20-base pair GC-rich region. Both subelements were required addi tively for transcription, but each element alone was sufficient for al most normal control by oxygen/heme deficiency, Mutations in both eleme nts decreased the induction ratio 3-4-fold. HEM13 UAS conferred a 2-4- fold oxygen/heme control on a heterologous reporter gene, Two Rox1p-re sponsive sites, R1 and R3, were identified, which accounted for the 6- 7-fold repression by Rox1p, A factor bound to a sequence close to site R3, This DNA-binding activity was only detected in protein extracts o f aerobic heme-sufficient ROX1 TUP1 cells, suggesting a possible role in site R3 function.