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<p>CIA5 is a zinc-finger-containing transcription regulator reported to be a master regulator of the critically important, inducible CO<sub>2</sub>-concentrating mechanism (CCM) of the model unicellular green alga, <i>Chlamydomonas</i>. Although mutants in the <i>CIA5</i> gene facilitated its identification more than two decades ago, we still know little about the detailed function of this important protein. Here, we report the first successful overexpression of full-length CIA5 protein in <i>E. coli</i>, confirmed by SDS-PAGE and Western immunoblots. We used these purified, full-length CIA5 proteins to identify potential specific DNA-binding sequences using random binding site selection (RBSS). This binding was confirmed using a gel mobility shift assay (GMSA) to demonstrate highly specific protein-DNA interactions 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 specific protein-DNA interaction with CIA5, although the GMSA interactions were somewhat weaker than with the RBSS-identified sequence. Nonetheless, this work provides the first direct evidence that CIA5 can bind specific DNA sequences in vitro, thus opening the way for more extensive in vivo experiments to determine the biological relevance of CIA5’s specific DNA-binding activity.</p>

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Investigation of the DNA Binding Ability of CIA5 in Chlamydomonas reinhardtii

  • 期刊论文

  • 发表日期：

    2026年1月21日

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  • Bo Chen,
  • Martin H. Spalding

摘要

CIA5 is a zinc-finger-containing transcription regulator reported to be a master regulator of the critically important, inducible CO2-concentrating mechanism (CCM) of the model unicellular green alga, Chlamydomonas. Although mutants in the CIA5 gene facilitated its identification more than two decades ago, we still know little about the detailed function of this important protein. Here, we report the first successful overexpression of full-length CIA5 protein in E. coli, confirmed by SDS-PAGE and Western immunoblots. We used these purified, full-length CIA5 proteins to identify potential specific DNA-binding sequences using random binding site selection (RBSS). This binding was confirmed using a gel mobility shift assay (GMSA) to demonstrate highly specific protein-DNA interactions 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 specific protein-DNA interaction with CIA5, although the GMSA interactions were somewhat weaker than with the RBSS-identified sequence. Nonetheless, this work provides the first direct evidence that CIA5 can bind specific DNA sequences in vitro, thus opening the way for more extensive in vivo experiments to determine the biological relevance of CIA5’s specific DNA-binding activity.