| Project/Area Number |
23770169
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| Research Category |
Grant-in-Aid for Young Scientists (B)
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| Allocation Type | Multi-year Fund |
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| Research Field |
Biophysics
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| Research Institution | Hokkaido University |
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Principal Investigator |
MIKUNI Shintaro 北海道大学, 大学院・医学研究科, 特任助教 (40435954)
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| Project Period (FY) |
2011 – 2012
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| Project Status |
Completed (Fiscal Year 2012)
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| Budget Amount *help |
¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
Fiscal Year 2012: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2011: ¥2,730,000 (Direct Cost: ¥2,100,000、Indirect Cost: ¥630,000)
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| Keywords | タンパク質 / 核酸の構造・動態・機能 / 転写因子 / バイオイメージング / 分子間相互作用 / 生物物理 / 核酸 / 蛋白質 / 転写活性 / 相関解析 / 相互作用 / 一分子計測 / FCS / FCCS / 核内受容体 / DNA |
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| Research Abstract |
Regulation of transcriptional activity could be initiated by associating the transcriptional factors with its specific sequence of genomic DNA. However, the direct relationship between “the affinity for DNA” and “transcriptional activity” of transcriptional factors is not clarified. To clarify this relationship, we tried to establish the simultaneous and quantitative assay for DNA binding and transcriptional activity of glucocorticoid receptor (GR), which is one of transcriptional factors, using fluorescence cross-correlation spectroscopy (FCCS). As a result, the dissociation constant between the purified GR fused with EGFP and the Alexa647-labeled GR specific sequences (glucocorticoid response element, GRE) could be determined as 0.63 μM, and sequence-specificity of GR could be confirmed by FCCS. Moreover, the dissociation constant between the purified RNA-specific binding protein fused withmCherry (red fluorescent protein) and Alexa488-labeled RNA was also determined as 143 nM by FCCS.
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