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Expression, purification and DNA-binding properties of zinc finger domains of DOF proteins from Arabidopsis thaliana.

ABSTRACT: Introduction: DOF proteins are a family of plant-specific transcription factors with a conserved zinc finger (ZF) DNA-binding domain. Although several studies have demonstrated their specific DNA binding, quantitative affinity data is not available for the binding of DOF domains to their binding sites. Methods: ZF domains of DOF2.1, DOF3.4, and DOF5.8 from Arabidopsis thaliana were expressed and purified. Their DNA binding affinities were assessed using gel retardation assays and microscale thermophoresis with two different oligonucleotide probes containing one and two copies of recognition sequence AAAG. Results: DOF zinc finger domains (DOF-ZFs) were shown to form independently folded structures. Assessments using microscale thermophoresis demonstrated that DOF-ZFs interact more tightly (~ 100 fold) with double-motif probe than the single-motif probe. The overall Kd values for the DOF3.4-ZF and DOF5.8-ZF to the double-motif probe were ~2.3±1 and 2.5±1 µM, respectively. Conclusion: Studied DOF-ZF domains formed stable complexes with the double-motif probe. Although DOF3.4-ZF and DOF5.8-ZF do not dimerize with an appreciable affinity in the absence of DNA (judging from size-exclusion and multiangle laser light scattering data), it is possible that these ZFs form protein-protein contacts when bound to this oligonucleotide, consistent with previous reports that DOF proteins can homo- and hetero-dimerize.

SUBMITTER: Moghaddas Sani H

PROVIDER: S-EPMC6128974 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Expression, purification and DNA-binding properties of zinc finger domains of DOF proteins from Arabidopsis thaliana.

Moghaddas Sani Hakimeh H Hamzeh-Mivehroud Maryam M Silva Ana P AP Walshe James L JL Mohammadi S Abolghasem SA Rahbar-Shahrouziasl Mahdyieh M Abbasi Milad M Jamshidi Omid O Low Jason Kk JK Dastmalchi Siavoush S Mackay Joel P JP

BioImpacts : BI 20180128 3

Introduction: DOF proteins are a family of plant-specific transcription factors with a conserved zinc finger (ZF) DNA-binding domain. Although several studies have demonstrated their specific DNA binding, quantitative affinity data is not available for the binding of DOF domains to their binding sites. Methods: ZF domains of DOF2.1, DOF3.4, and DOF5.8 from Arabidopsis thaliana were expressed and purified. Their DNA binding affinities were assessed using gel ret ...[more]

PMID: 30211076

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Project description:BackgroundThe classical C2H2 zinc finger domain is involved in a wide range of functions and can bind to DNA, RNA and proteins. The comparison of zinc finger proteins in several eukaryotes has shown that there is a lot of lineage specific diversification and expansion. Although the number of characterized plant proteins that carry the classical C2H2 zinc finger motifs is growing, a systematic classification and analysis of a plant genome zinc finger gene set is lacking.ResultsWe found through in silico analysis 176 zinc finger proteins in Arabidopsis thaliana that hence constitute the most abundant family of putative transcriptional regulators in this plant. Only a minority of 33 A. thaliana zinc finger proteins are conserved in other eukaryotes. In contrast, the majority of these proteins (81%) are plant specific. They are derived from extensive duplication events and form expanded families. We assigned the proteins to different subgroups and families and focused specifically on the two largest and evolutionarily youngest families (A1 and C1) that are suggested to be primarily involved in transcriptional regulation. The newly defined family A1 (24 members) comprises proteins with tandemly arranged zinc finger domains. Family C1 (64 members), earlier described as the EPF-family in Petunia, comprises proteins with one isolated or two to five dispersed fingers and a mostly invariant QALGGH motif in the zinc finger helices. Based on the amino acid pattern in these helices we could describe five different signature sequences prevalent in C1 zinc finger domains. We also found a number of non-finger domains that are conserved in these families.ConclusionsOur analysis of the few evolutionarily conserved zinc finger proteins of A. thaliana suggests that most of them could be involved in ancient biological processes like RNA metabolism and chromatin-remodeling. In contrast, the majority of the unique A. thaliana zinc finger proteins are known or suggested to be involved in transcriptional regulation. They exhibit remarkable differences in the features of their zinc finger sequences and zinc finger arrangements compared to animal zinc finger proteins. The different zinc finger helix signatures we found in family C1 may have important implications for the sequence specific DNA recognition and allow inferences about the evolution of the members in this family.

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Expression, purification and DNA-binding properties of zinc finger domains of DOF proteins from Arabidopsis thaliana.

Publications

Expression, purification and DNA-binding properties of zinc finger domains of DOF proteins from <i>Arabidopsis thaliana</i>.

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