title: Electrophoretic mobility shift assay reveals a novel recognition sequence for Setaria italica NAC protein.
abstract: The NAC (NAM/ATAF1,2/CUC2) proteins are among the largest family of plant transcription factors. Its members have been associated with diverse plant processes and intricately regulate the expression of several genes. Inspite of this immense progress, knowledge of their DNA-binding properties are still limited. In our recent publication,1 we reported isolation of a membrane-associated NAC domain protein from Setaria italica (SiNAC). Transactivation analysis revealed that it was a functionally active transcription factor as it could stimulate expression of reporter genes in vivo. Truncations of the transmembrane region of the protein lead to its nuclear localization. Here we describe expression and purification of SiNAC DNA-binding domain. We further report identification of a novel DNA-binding site, [C/G][A/T][T/A][G/C]TC[C/G][A/T][C/G][G/C] for SiNAC by electrophoretic mobility shift assay. The SiNAC-GST protein could bind to the NAC recognition sequence in vitro as well as to sequences where some bases had been reshuffled. The results presented here contribute to our understanding of the DNA-binding specificity of SiNAC protein.
authors.affiliation: National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, India.
authors.forename: Swati
authors.initials: S
authors.lastname: Puranik
authors.forename: Karunesh
authors.initials: K
authors.lastname: Kumar
authors.forename: Prem S
authors.initials: PS
authors.lastname: Srivastava
authors.forename: Manoj
authors.initials: M
authors.lastname: Prasad
mesh_terms: D001483:Base Sequence; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D032983:Setaria Plant / Q000378:metabolism*
chemical_list: D010940:Plant Proteins