鉴定水稻中一个新的专一结合GCC元件的AP2/EREBP族转录因子

Characterization of a Novel AP2/EREBP Transcription Factor TSH1 Specifically Binding to GCC Elements

TSH1,是通过搜寻GenBank的EST库而获得的一个来源于水稻的含AP2/EREBP保守结构域的蛋白质.为了详细分析TSH1蛋白与其DNA顺式元件的结合特性,首先采用传统的凝胶阻滞实验和酵母单杂交技术,证实TSH1在体内和体外均专一性地结合于GCC元件,然后利用原子力显微镜技术精确测量了TSH1蛋白与GCC元件在单分子水平的相互作用力.结果表明,GST-TSH1与DRE元件没有特异性的结合,而GST-TSH1与GCC元件结合力的大小为(83.9±2.2)pN,这种特异性的结合可以被加入的游离TSH1蛋白明显降低.GST蛋白和突变GCC元件作为负对照显示出与GCC元件无特异性作用力.以上结果充分证明,TSH1是专一性地与GCC元件相作用的转录因子,而且原子力显微镜对于检测转录因子与DNA相互作用时单碱基的突变十分灵敏.通过比较几种评估转录因子与DNA顺式元件结合特异性的方法,阐述了原子力显微镜技术的特点及优越性.

TSH1, a novel transcription factor in rice containing conserved AP2/EREBP domain, was screened from the EST database. The specific interaction of TSH1 and DNA elements was detected by two kinds of traditional methods, electronic mobility assay and yeast one-hybrid assay, which suggested that THS1 specifically binds to GCC element in vitro and in vivo. Furthermore, It was confirmed by atom force microscopy （AFM） that TSH1 binds to GCC element （core sequence GCCGCC） not DRE element （core sequence GCCGAC）, and the single molecular force between TSH1 and GCC element was quantitatively measured. The molecular force of TSH1 with GCC element is （83.9 ± 2.2） pN. This interaction can be observably blocked by the dissociate TSH1 protein in the solution. These above results proved that TSH1 recognizes and binds to GCC with specifically interaction, which also demonstrate the high sensitivity of the AFM measurements for the single base substitution of the GCC core sequence greatly reduced the binding affinity. Comparing these methods for identification the binding of transcription factor with its DNA element, it was considered that AFM is expected to be a sensitive and reliable method that offers valuable information for the characterization of transcription factors.