Solution structure of GCCAAT recognition motif by 2D NMR, spectral simulation, molecular modeling, and distance geometry calculations.
Solution structure of GCCAAT recognition motif by 2D NMR, spectral simulation, molecular modeling, and distance geometry calculations.
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通过 2D NMR、光谱模拟、分子建模和距离几何计算得出 GCCAAT 识别基序的溶液结构。
DOI：
10.1021/bi00086a010
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Virander S. Chauhan
中科院分区：
文献类型：
--
作者：
R. Nibedita;R. A. Kumar;A. Majumdar;R. Hosur;G. Govil;K. Majumder;Virander S. Chauhan
用核磁共振波谱研究了含有几种转录因子的GCCAAT识别基序的自互补14聚体DNA双链(d-GGATTGGCCAATCC)的溶液构象。所有的质子(除了H5‘，H5’‘质子)都按照基于二维核磁共振技术的标准程序进行了完全的共振归属。用光谱模拟方法确定了三键耦合常数。从结构的角度描述和采用了新的策略来量化NOE强度。通过对谱的H_1‘-H_4’交叉峰的J(4‘-5’)、J(4‘-5’)或J(4‘-5’)之和的估计，从选择性的NOESY实验得到了伽马扭角的近似范围。同样，通过监测选择性NOESY谱中H8/H6-H3‘交叉峰中的H3’重数，得到了epsilon扭角的范围。在73个耦合约束、79个NOE强度约束和108个氢键约束的帮助下，进行了建模，得到了满足约束的结构。从这样的结构出发，创建了扩展的距离约束集，该约束集已用于使用程序Tandy进行距离几何计算。在这样得到的最佳结构中，在中心观察到了类似于BI-BII转变的有趣的不规则性。该分子呈弯曲状。总体碱基堆积与B-DNA或A-DNA模型中的不同。碱基对相对于局部螺旋轴倾斜。观察到的结构特征可能对GCCAAT基序的识别机制有重要影响。
Solution conformation of a self-complementary 14-mer DNA duplex (d-GGATTGGCCAATCC) containing the GCCAAT recognition motif of several transcription factors has been investigated by NMR spectroscopy. Complete resonance assignment of all the protons (except H5',H5'' protons) has been obtained following standard procedures based on two-dimensional NMR techniques. Three-bond coupling constants have been determined by spectral simulation procedures. New strategies have been described and employed for quantifying NOE intensities from the structural point of view. Approximate ranges of gamma torsion angles have been obtained from a selective NOESY experiment, by estimating the J(4'-5'), J(4'-5''), or their sum in the H1'-H4' cross peaks of the spectrum. Likewise, ranges of epsilon torsion angles have been obtained by monitoring the H3' multiplicities in the H8/H6-H3' cross peaks in selective NOESY spectra. With the help of such a total of 73 coupling constraints, 79 NOE intensity constraints, and 108 H-bond constraints, model building has been carried out to obtain a structure which satisfies the constraints. Starting from such a structure, an expanded distance constraint set has been created which has been used for the distance geometry calculations using the program TANDY. In the best structure thus derived, interesting irregularities similar to a BI-BII transition have been observed in the center. The molecule exhibits a bend. The overall base stacking is different from that in either B- or A-DNA models. The base pairs are tilted with respect to the local helix axes. The observed structural features are likely to have important implications for the recognition mechanism of the GCCAAT motif.