Study on the molecular mechanism of Arabidopsis PIF3 binding to G-box cis-elements

• Main Authors: Chia-Yu Chien, 簡嘉佑
• Other Authors: Yi-Sheng Cheng
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/97630203549687728927

碩士 === 國立臺灣大學 === 植物科學研究所 === 102 === It is crucial for plants to sense light signals by phytochromes in development and growth. Among phytochrome signaling pathway, PHYTOCHROME INTERACTING FACTOR3 (PIF3) is believed to act as a key component that negatively regulates several light responses in plants. Previous studies have identified PIF3 as a member of basic helix-loop-helix (bHLH) transcription factor superfamily that can directly bind to a typical G-box (CACGTG) DNA. In this study, we aimed to reveal the binding mechanism between PIF3 and G-box DNA from structural view point. In this study, we examined the oligomeric states of apo form bHLH and bHLH-DNA by gel filtration chromatography and analytical ultracentrifugation. The results indicated that bHLH might form a homodimer to associate with G-box DNA. We also found some hydrophobic residues, including Met60, Val57, Gln56, Leu53, Leu50, Tyr49, Ile47, Leu27 and Met24 by sequence alignment and modellng. These residues formed a six-layer hydrophobic interaction interface that might be critical for bHLH dimer formation. From the fluorescein-based electrophoretic mobility shift assay (fEMSA) results, we found that not only G-box but also E-box (CANNTG) could bind to bHLH protein. In addition, we found some conserved residues His9, Glu13 and Arg17 that might play critical roles in G-box recognition. Combined with fEMSA and modelling results, the Arg17 that formed hydrogen bonds with the 4th base of G-box may not act as a DNA recognition residue. Although the 3th base of G-box did not participate in hydrogen bond formation, it might have steric hindrance that affected the hydrogen bond formation between Arg17 and the 4th base of G-box. In summary, we concluded a possible mechanism of bHLH binding to G-box sequence: The bHLH formed homodimers by the hydrophobic residues first. The positive-charged binding groove of bHLH could stabilize DNA at the groove. Then, the His9 and Glu13 could recognize the 6th base of G-box and the the 2th base of anti-sense G-box. The Arg17 then formed hydrogen bonds with the 4th base of G-box. Finally, they could form a hydrogen bond network to stabilize the protein-DNA structure.