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 synthesize d 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 transcripti on, with no contribution from post-transcriptional processes. To under stand the mechanism underlying its regulation, the genomic DNA encodin g cyt c(6) (Cyc6) was analyzed for the presence of copper-responsive e lements. 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 be ta-tubulin promoter-driven (arylsulfatase-encoding) reporter gene. Ana lysis of this 120-bp fragment indicated that copper-responsive element s 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-resp onsive pathway. In the context of a constitutive promoter (derived fro m the beta-tubulin gene), each region was found to function as an acti vator of transcription in copper-deficient cells, and the metal specif icity 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' up stream sequences.