The flanking sequence of W-box influences the binding of Arabidopsis WRKY54 DNA binding domain
Date Issued
2016
Date
2016
Author(s)
Hsieh, Min-Che
Abstract
WRKY transcription factors are exclusive in plants and involved in biotic and abiotic stresses. Arabidopsis WRKY54(AT2G40750) belongs to Group III of the WRKY family and consists of a WRKY domain with zinc-finger of C2HC that specifically binds to W box (TTGACC/T) in plant genome. WRKY54 involves in salicylic acid signal transduction and could respond to system defense in plants. It is unclear if the flanking sequence of W-box could affect the binding specificity of WRKY transcription factor. In this study, we aim to find out the influence of the flanking sequence on binding specificity of WRKY transcription factor. Using affinity chromatography and size exclusion chromatography, we successfully purified WRKY54 DNA binding domain(DBD) recombinant protein. Dynamic light scattering identified that WRKY54 DBD recombinant protein is a monomer and mono-dispersed in solution and its molecular weight is 59 kDa. Fluorescein-based electrophoretic mobile shift assay (fEMSA) and Quartz-crystal microbalance (QCM) have confirmed WRKY54 could bind to various W-boxes DNA fragments and the results indicated that flanking region of W-box would affect the binding affinity of WRKY54. SAG12 is a gene to induce leaf senescence, its promoter consists of four W-boxes and only W4 can be recognized in ChIP(chromatin immno-preicpitation) assay. It is also confirmed that the flanking sequence of W-box plays an important role for WRKY54 transcription factor. Based on structural modeling of WRKY54 and W-box, we proposed that the binding residues of WRKY54 would recognize W-box with its flanking region (TxTTGACT). This study could reveal the binding specificity between WRKY transcription factors and W-box for further study.
Subjects
WRKY54 DNA binding domain
Transcription factor
W-box binding specificity
Type
thesis
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