Lorenz Mike, Hillisch Alexander, Diekmann Stephan
Institut für Molekulare Biotechnologie, Jena, Germany.
J Biotechnol. 2002 Jan;82(3):197-209. doi: 10.1016/s1389-0352(01)00038-1.
Fluorescence resonance energy transfer studies allow to determine global shape properties of nucleic acids and nucleoprotein complexes. In many DNA-protein complexes, the DNA is more or less bent and the degree of bending can be obtained by FRET. For example, the DNA in complex with the integration host factor (IHF) is kinked by approximately 160 degrees building a U-shaped structure. The two DNA helix ends come close to one another in space in a distance range easily measurable by FRET. The global DNA structure of this complex can be mimicked by introducing two regions with unpaired bases ('bulges') into the DNA each producing a sharp kink of approximately 80 degrees. These U-shaped DNA constructs were used to measure the electrostatic interaction of the two nearly parallel negatively charged DNA helix arms. The electrostatic repulsion between the helix arms, and as a consequence their distance, decreases with growing salt concentration of mono- or divalent cations. This experimental approach also allows the sensitive study of the local structure of DNA sequences positioned between the two bulges.
荧光共振能量转移研究有助于确定核酸和核蛋白复合物的整体形状特性。在许多DNA-蛋白质复合物中，DNA或多或少会发生弯曲，弯曲程度可通过荧光共振能量转移获得。例如，与整合宿主因子（IHF）结合的DNA会发生约160度的扭结，形成U形结构。两条DNA螺旋末端在空间中彼此靠近，其距离范围可通过荧光共振能量转移轻松测量。通过在DNA中引入两个带有未配对碱基（“凸起”）的区域，每个区域产生约80度的急剧扭结，可模拟该复合物的整体DNA结构。这些U形DNA构建体用于测量两条近乎平行的带负电荷的DNA螺旋臂之间的静电相互作用。螺旋臂之间的静电排斥以及由此导致的它们之间的距离，会随着单价或二价阳离子盐浓度的增加而减小。这种实验方法还允许对位于两个凸起之间的DNA序列的局部结构进行灵敏研究。
