IN-VITRO BINDING OF NUCLEAR PROTEINS TO THE BARLEY PLASTOCYANIN GENE PROMOTER REGION

Plastocyanin is a nuclear-encoded chloroplast protein participating in electron transport during photosynthesis. The plastocyanin gene is expressed in photosynthetic tissue in a developmentally regulated manner and the expression is stimulated by light. A genomic clone encoding the plastocyanin precursor was isolated from a barley (Hordeum vulgare) lambda library using a barley cDNA clone as a probe and the sequence of a 1.9-kb DNA fragment containing the plastocyanin gene was determined. TATA and CCAAT boxes are located 34-bp and 68-bp, respectively, upstream of the transcription start site, the 5'-untranslated leader is 78 nucleotides long, and the intronless gene has at least two different polyadenylation sites. DNA sites in the plastocyanin gene that mediate binding of barley nuclear proteins were mapped by mobility-shift assays with fragments of the promoter/upstream region. Two of the three specific binding sites characterised in more detail were found to form complexes with the same factor in cross-competition experiments. One of these sites, narrowed down to a 17-bp sequence at position -512, contains the consensus binding site for Myb-like transcription factors. The third specific binding site, located at position -622, contains the sequence CACGTG which is a high-affinity-binding site for transcription factors of the basic-region leucine-zipper family.