2 COPPER-RESPONSIVE ELEMENTS ASSOCIATED WITH THE CHLAMYDOMONAS CYC6 GENE-FUNCTION AS TARGETS FOR TRANSCRIPTIONAL ACTIVATORS

In Chlamydomonas reinhardtii, cytochrome c(6) (cyt c(6)) is synthesized only under conditions of copper deficiency when plastocyanin cannot be synthesized. In previous work, the copper-responsive regulation of cyt c(6) 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 c(6) (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 c(6) 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 c(6) is thus attributed to these two 5' upstream sequences.