Tonelli M, Ragg E, Bianucci A M, Lesiak K, James T L
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446, USA.
Biochemistry. 1998 Aug 25;37(34):11745-61. doi: 10.1021/bi980481n.
The three-dimensional structure of d(GCATATGATAG).d(CTATCATATGC), from the promoter region of a gene regulating sporulation in Bacillus subtilis mother cells, was determined utilizing two-dimensional nuclear Overhauser effect (2D NOE) and double-quantum-filtered COSY (2QF-COSY) spectra. To minimize the effect of methods used to obtain restraints and refine structure, several variables were studied. Interproton distance bounds were calculated very conservatively by running the complete relaxation matrix program MARDIGRAS hundreds of times using 2D NOE spectra for exchangeable and for nonexchangeable protons at different mixing times, assuming different overall correlation times and different starting structures. The 435 distance restraints were used with two different structural refinement methods: restrained molecular dynamics (rMD) and restrained Monte Carlo calculations (rMC). Refinement using different procedures and starting structures resulted in essentially the same structure (<0.8 A rmsd), indicating that the structure is defined by experimental restraints and not the refinement method or variables used. R factors indicate the structures fit the experimental NOE data very well. Some helical parameters, notably large negative X displacement, are characteristic of A-DNA, but others are characteristic of B-DNA. As with TG.CA steps in other duplex DNA sequences studied in our laboratory, the two TG.CA steps have a positive roll, with T6-G7 exhibiting the largest, and consequently a bent helix axis. The converged structure represents a time-averaged structure. However, multiple conformations, especially in deoxyriboses, were evident from vicinal coupling constants obtained from quantitative simulations of 2QF-COSY cross-peaks and from persistent inconsistencies in experimental distances due to nonlinear conformational averaging.
利用二维核Overhauser效应（2D NOE）和双量子滤波COSY（2QF - COSY）光谱，确定了来自枯草芽孢杆菌母细胞中调节孢子形成的基因启动子区域的d(GCATATGATAG).d(CTATCATATGC)的三维结构。为了尽量减少用于获得约束和优化结构的方法的影响，研究了几个变量。通过在不同混合时间使用可交换和不可交换质子的2D NOE光谱运行完整的弛豫矩阵程序MARDIGRAS数百次，假设不同的整体相关时间和不同的起始结构，非常保守地计算了质子间距离边界。435个距离约束与两种不同的结构优化方法一起使用：约束分子动力学（rMD）和约束蒙特卡罗计算（rMC）。使用不同程序和起始结构进行优化得到的结构基本相同（均方根偏差<0.8 Å），这表明该结构由实验约束定义，而非优化方法或所使用的变量。R因子表明这些结构与实验NOE数据拟合得非常好。一些螺旋参数，特别是大的负X位移，是A - DNA的特征，但其他一些则是B - DNA的特征。与我们实验室研究的其他双链DNA序列中的TG.CA步一样，这两个TG.CA步具有正向滚动，其中T6 - G7的滚动最大，因此螺旋轴弯曲。收敛结构代表一个时间平均结构。然而，从2QF - COSY交叉峰的定量模拟获得的邻位耦合常数以及由于非线性构象平均导致的实验距离的持续不一致中可以明显看出存在多种构象，尤其是在脱氧核糖中。
