Single molecule study of binding force between transcription factorTINY and its DNA responsive element

The interaction force between transcription factor TINY and its corresponding cis-element has been studied with single molecule force microscopy. TINY belongs to the AP2/EREBP family, which is the second largest transcription factor family in plant and plays an important role in the genetic regulation of plant development and growth. The results showed that TINY bound to dehydration-responsive element (DRE) with the core sequence A/GCCGAC efficiently. The single molecule forces between TINY and DRE A/GCCGAC measured by atomic force microscopy were 83.5±3.4 and 81.4±4.9 pN, respectively. Either the single base substitution of the DRE core sequence or point mutation of the key amino acid in TINY DNA-binding domain greatly reduced the binding strength. We have previously reported the method of using AFM to measure the interaction force between transcription factor ZmDREB1A, another AP2/EREBP family protein, and DRE responsive element. The results in this work further confirm the applicability and high sensitivity of AFM measurements in transcription factor-DNA interaction study. More interesting, it was found that 19th amino acid mutation of TINY resulted in the decrease of the binding force but the binding probability was the same as the wild type TINY. The reduced binding force was correlated with the reduced activity of TINY regulated reporter gene in the yeast one-hybrid experiment. It is thus expected that the single molecule force measurement offers valuable information on the analysis of transcription factor regulated gene expression.