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Volume 271, Number 40, Issue of October 4, 1996 pp. 24425-24432
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Characterization of an Upstream Activation Sequence and Two Rox1p-responsive Sites Controlling the Induction of the Yeast HEM13 Gene by Oxygen and Heme Deficiency

(Received for publication, March 7, 1996, and in revised form, June 14, 1996)

From the Laboratoire de Biochimie des Porphyrines, Institut Jacques Monod, Université Paris VII, 2 Place Jussieu, 75251 Paris, France

The Saccharomyces cerevisiae HEM13 gene codes for coproporphyrinogen oxidase, an oxygen-requiring enzyme catalyzing the sixth step of heme biosynthesis. Its transcription has been shown to be induced 40-50-fold in response to oxygen or heme deficiency, in part through relief of repression exerted by Rox1p and in part by activation mediated by an upstream activation sequence (UAS). This report describes an analysis of HEM13 UAS and of the Rox1p-responsive sites by electrophoretic mobility shift assays, DNase I footprinting, and mutational mapping. HEM13 UAS is composed of two subelements: a 16-base pair sequence binding a constitutive factor acting as a transcriptional activator, and a 5-flanking 20-base pair GC-rich region. Both subelements were required additively for transcription, but each element alone was sufficient for almost normal control by oxygen/heme deficiency. Mutations in both elements decreased the induction ratio 3-4-fold. HEM13 UAS conferred a 2-4-fold oxygen/heme control on a heterologous reporter gene. Two Rox1p-responsive 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 of aerobic heme-sufficient ROX1 TUP1 cells, suggesting a possible role in site R3 function.

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