A Repressor with Similarities to Prokaryotic and Eukaryotic DNA Helicases Controls the Assembly of the CAAT Box Binding Complex at a Photosynthesis Gene Promoter

Abstract

A single nucleotide exchange in a promoter region located immediately upstream of the CAAT box of the spinach photosynthesis gene AtpC (gene product is sub-unit γ of the chloroplast ATP synthase) prevents the formation of a secondary structure and causes an unregulated, constitutive high level of expression (Kusnetsov, V., Landsberger, M., Oelmüller, R. (1999) J. Biol. Chem. 274, 36009-36014). We have isolated cDNAs for ATPC-2, a new polypeptide with homologies to pro- and eukaryotic helicases, which specifically binds to this promoter region. Binding of ATPC-2 competes strongly with that of a CAAT box binding factor (CBF), consistent with the idea that both complexes cannot be formed simultaneously because of sterical reasons. In gel mobility shift assays, high binding activities of ATPC-2 and low binding activities of CBF were observed with nuclear extracts from tissue with low AtpC expression levels, and the opposite was observed with extracts from tissues with high AtpC expression levels. Binding of ATPC-2 to the mutant sequence, which directs a constitutively high level expression in vivo and prevents the formation of a secondary structure in vitro, is significantly weaker than binding to the wild-type sequence. Again, the opposite results were obtained for the CBF. Thus, we conclude that the assembly of the CBF·DNA complex stimulates transcription of AtpC and that CBF binding is prevented if ATPC-2 is bound to the promoter region. The novel mechanism of gene regulation and the role of the helicase-like protein ATPC-2 as a potential transcriptional repressor is discussed in relation to its modular structure.