DNA-protein interactions throughout the Arabidopsis calmodulin-3 gene
Szymanski, Daniel Benedict
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Permalink
https://hdl.handle.net/2142/22702
Description
Title
DNA-protein interactions throughout the Arabidopsis calmodulin-3 gene
Author(s)
Szymanski, Daniel Benedict
Issue Date
1995
Doctoral Committee Chair(s)
Zielinski, Raymond E.
Department of Study
Plant Biology
Discipline
Plant Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Biology, Botany
Chemistry, Biochemistry
Language
eng
Abstract
In Arabidopsis thaliana, calmodulin is encoded by a gene family of at least six members that generate four unique, but highly similar calmodulin polypeptides. In vitro techniques were used to identify putative cis-acting elements in the calmodulin-3 gene and the types of nuclear factors with which they interact with the goal of defining the molecular mechanisms regulating calmodulin-3 transcription. The $-$685 to $-$385 region of the calmodulin-3 promoter interacts with abundant A/T-sequence binding nuclear factors, termed high mobility group proteins, which are thought to play an architectural role in regulating the chromatin structure of a gene. Sequence specific DNA-binding proteins that recognize sequence elements in the 5$\prime$-flanking, intron, and 3$\prime$ untranslated region of the calmodulin-3 gene were also detected. One recognition element centered on bp $-$260 contains numerous TGAC-repeats, and is similar to well characterized Agrobacterium T-DNA and viral activating promoter elements (nos, as-1) and conserved elements in auxin induced genes in Arabidopsis and Glycine max. Fusions between the calmodulin-3 promoter and the $\beta$-glucuronidase reporter gene were introduced into tobacco plants to the biological roles of the protein binding sites identified in vitro. Consistent with activity of similar sequence elements in the cauliflower mosaic virus 35S promoter, the TGAC-repeat domain of the calmodulin-3 promoter specifically activated reporter protein expression in roots. The TGAC-repeat domain was recognized by a family of at least six TGA-like factors in cauliflower nuclear extract and recombinant Arabidopsis TGA3. TGA-related factor binding to the CaM-3 promoter was inhibited by nuclear factors that bind a downstream binding site centered on bp $-$225. Western blot data and amino acid sequence comparisons showed that the plant TGA-like factors are similar to the regulatory domains of the vertebrate cAMP responsive element binding protein, CREB. DNA-binding of TGA-like factors in cauliflower nuclear extract and recombinant Arabidopsis TGA3 are subject to post-translational modification by calmodulin. The calmodulin stimulation of DNA-binding in mobility shift assays was Ca$\sp{2+}$-dependent and isoform specific. Calmodulin overlay experiments showed that biotinylated calmodulin interacts with TGA3 and factors in cauliflower nuclear extract in the absence of DNA in a Ca$\sp{2+}$-dependent manner. These results have potential significance in understanding the integration of Ca$\sp{2+}$ signaling events with transcriptional activation of specific promoters.
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