Isolation of a calmodulin-binding transcription factor from rice (Oryza sativa L.).

J Biol Chem

Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju 660-701, Korea.

Published: December 2005

AI Article Synopsis

  • Calmodulin (CaM) is involved in regulating various cellular functions, and this study identifies a new transcription factor, OsCBT, in rice that binds to CaM.
  • OsCBT has a distinct structure with a DNA-binding domain and multiple CaM-binding motifs and demonstrates specific DNA binding.
  • The study reveals that OsCBT activates gene expression but its function is inhibited when CaM is present, suggesting a complex regulatory role for CaM in transcription.

Article Abstract

Calmodulin (CaM) regulates diverse cellular functions by modulating the activities of a variety of enzymes and proteins. However, direct modulation of transcription factors by CaM has been poorly understood. In this study, we isolated a putative transcription factor by screening a rice cDNA expression library by using CaM:horse-radish peroxidase as a probe. This factor, which we have designated OsCBT (Oryza sativa CaM-binding transcription factor), has structural features similar to Arabidopsis AtSRs/AtCAMTAs and encodes a 103-kDa protein because it contains a CG-1 homology DNA-binding domain, three ankyrin repeats, a putative transcriptional activation domain, and five putative CaM-binding motifs. By using a gel overlay assay, gel mobility shift assays, and site-directed mutagenesis, we showed that OsCBT has two different types of functional CaM-binding domains, an IQ motif, and a Ca(2+)-dependent motif. To determine the DNA binding specificity of OsCBT, we employed a random binding site selection method. This analysis showed that OsCBT preferentially binds to the sequence 5'-TWCG(C/T)GTKKKKTKCG-3' (W and K represent A or C and T or G, respectively). OsCBT was able to bind this sequence and activate beta-glucuronidase reporter gene expression driven by a minimal promoter containing tandem repeats of these sequences in Arabidopsis leaf protoplasts. Green fluorescent protein fusions of two putative nuclear localization signals of OsCBT, a bipartite and a SV40 type, were predominantly localized in the nucleus. Most interestingly, the transcriptional activation mediated by OsCBT was inhibited by co-transfection with a CaM gene. Taken together, our results suggest that OsCBT is a transcription activator modulated by CaM.

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http://dx.doi.org/10.1074/jbc.M504616200DOI Listing

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