Abstract
CIA5 is a zinc-finger containing transcription regulator reported to be a master regulator of the critically important, inducible CO2-concentrating mechanism of the model, unicellular green alga, Chlamydomonas. Although mutants in the CIA5 gene facilitated identification of CIA5 more than two decades ago, we still know little about the detailed function of this important protein. Here we report the first successful over-expression of full length CIA5 proteins in E. coli, confirmed by SDS-PAGE and Western immunoblots. We also used these purified, full length CIA5 proteins to identify potential specific DNA-binding sequences using random binding site selection (RBSS), which was confirmed using a gel mobility shift assay (GMSA) to demonstrate highly specific protein-DNA interaction with purified, full-length CIA5. In addition, we identified a 9-bp GC rich (GGGGCGGGG) motif from the promoters of CIA5 dependent genes, and demonstrated using GMSA that promoter fragments containing this candidate motif from three CIA5-dependent genes also showed highly specific protein-DNA interaction with CIA5, although the GMSA interactions were somewhat weaker than with the RBSS-identified sequence. Nonetheless, this work clearly provides the first direct evidence that CIA5 can bind specific DNA sequences in vitro and thus opens the way for more extensive in vivo experiments to determine whether the specific DNA-binding of CIA5 has any biological relevance in vivo.