title: Two copper-responsive elements associated with the Chlamydomonas Cyc6 gene function as targets for transcriptional activators.
abstract: In Chlamydomonas reinhardtii, cytochrome c6 (cyt c6) is synthesized only under conditions of copper deficiency when plastocyanin cannot be synthesized. In previous work, the copper-responsive regulation of cyt c6 synthesis was demonstrated to occur by control of transcription, with no contribution from post-transcriptional processes. To understand the mechanism underlying its regulation, the genomic DNA encoding cyt c6 (Cyc6) was analyzed for the presence of copper-responsive elements. Sequences lying between positions -127 and -7 with respect to the start site of transcription were found to be sufficient to confer copper-responsive expression on either a promoterless or a minimal beta-tubulin promoter-driven (arylsulfatase-encoding) reporter gene. Analysis of this 120-bp fragment indicated that copper-responsive elements lie in two distinct regions (between -110 to -56 and -127 to -109). ATG fusions between copper-insensitive promoters and the coding plus 3' untranslated region of the Cyc6 gene resulted in the accumulation of cyt c6 in copper-supplemented medium; this confirms earlier studies indicating a lack of post-transcriptional control in this copper-responsive pathway. In the context of a constitutive promoter (derived from the beta-tubulin gene), each region was found to function as an activator of transcription in copper-deficient cells, and the metal specificity of the response of reporter genes containing either one or both regions was identical to that of the endogenous Cyc6 gene. The copper-responsive synthesis of cyt c6 is thus attributed to these two 5' upstream sequences.
authors.affiliation: Department of Chemistry and Biochemistry, University of California at Los Angeles 90095-1569, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Quinn
authors.forename: S
authors.initials: S
authors.lastname: Merchant
mesh_terms: D000818:Animals; D001192:Arylsulfatases / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D003300:Copper / Q000494:pharmacology*; D003573:Cytochrome b Group / Q000096:biosynthesis / Q000235:genetics*; D045346:Cytochrome b6f Complex; D004252:DNA Mutational Analysis; D018506:Gene Expression Regulation, Plant / Q000187:drug effects*; D017343:Genes, Plant / Q000235:genetics; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014170:Transformation, Genetic
chemical_list: D003573:Cytochrome b Group; D014157:Transcription Factors; D003300:Copper; D045346:Cytochrome b6f Complex; D001192:Arylsulfatases