Ojha R P, Dhingra M M, Sarma M H, Myer Y P, Setlik R F, Shibata M, Kazim A L, Ornstein R L, Rein R, Turner C J, Sarma R H
Department of Chemistry, University at Albany, NY 12222, USA.
J Biomol Struct Dyn. 1997 Oct;15(2):185-215. doi: 10.1080/07391102.1997.10508186.
The structure of an anti-HIV-1 ribozyme-DNA abortive substrate complex was investigated by 750 MHz NMR and computer modeling experiments. The ribozyme was a chimeric molecule with 30 residues-18 DNA nucleotides, and 12 RNA residues in the conserved core. The DNA substrate analog had 17 residues. The chimeric ribozyme and the DNA substrate formed a shortened ribozyme-abortive substrate complex of 47 nucleotides with two DNA stems (stems I and III) and a loop consisting of the conserved core residues. Circular dichroism spectra showed that the DNA stems assume A-family conformation at the NMR concentration and a temperature of 15 degrees C, contrary to the conventional wisdom that DNA duplexes in aqueous solution populate entirely in the B-form. It is proposed that the A-family RNA residues at the core expand the A-family initiated at the core into the DNA stems because of the large free energy requirement for the formation of A/B junctions. Assignments of the base H8/H6 protons and H1' of the 47 residues were made by a NOESY walk. In addition to the methyl groups of all T's, the imino resonances of stems I and III and AH2's were assigned from appropriate NOESY walks. The extracted NMR data along with available crystallographic data, were used to derive a structural model of the complex. Stems I and III of the final model displayed a remarkable similarity to the A form of DNA; in stem III, a GC base pair was found to be moving into the floor of the minor groove defined by flanking AT pairs; data suggest the formation of a buckled rhombic structure with the adjacent pair; in addition, the base pair at the interface of stem III and the loop region displayed deformed geometry. The loop with the catalytic core, and the immediate region of the stems displayed conformational multiplicity within the NMR time scale. A catalytic mechanism for ribozyme action based on the derived structure, and consistent with biochemical data in the literature, is proposed. The complex between the anti HIV-1 gag ribozyme and its abortive DNA substrate manifests in the detection of a continuous track of A.T base pairs; this suggests that the interaction between the ribozyme and its DNA substrate is stronger than the one observed in the case of the free ribozyme where the bases in stem I and stem III regions interact strongly with the ribozyme core region (Sarma, R. H., et al. FEBS Letters 375, 317-23, 1995). The complex formation provides certain guidelines in the design of suitable therapeutic ribozymes. If the residues in the ribozyme stem regions interact with the conserved core, it may either prevent or interfere with the formation of a catalytically active tertiary structure.
通过750兆赫核磁共振（NMR）和计算机模拟实验研究了抗HIV - 1核酶 - DNA流产底物复合物的结构。该核酶是一种嵌合分子，有30个残基——18个DNA核苷酸，以及12个位于保守核心区的RNA残基。DNA底物类似物有17个残基。嵌合核酶和DNA底物形成了一个47个核苷酸的缩短的核酶 - 流产底物复合物，有两个DNA茎（茎I和茎III）以及一个由保守核心残基组成的环。圆二色光谱表明，在核磁共振浓度和15摄氏度的温度下，DNA茎呈现A族构象，这与传统观点认为水溶液中的DNA双链完全以B型存在相反。有人提出，核心区的A族RNA残基由于形成A/B连接所需的大量自由能，将在核心区起始的A族扩展到DNA茎中。通过NOESY步移对47个残基的碱基H8/H6质子和H1'进行了归属。除了所有T的甲基，茎I和茎III的亚氨基共振以及AH2也通过适当的NOESY步移进行了归属。提取的核磁共振数据与现有的晶体学数据一起用于推导复合物的结构模型。最终模型的茎I和茎III与DNA的A形式有显著相似性；在茎III中，发现一个GC碱基对移动到由侧翼AT对定义的小沟底部；数据表明与相邻对形成了一个弯曲的菱形结构；此外，茎III与环区域界面处的碱基对显示出变形的几何形状。带有催化核心的环以及茎的紧邻区域在核磁共振时间尺度内表现出构象多样性。基于推导结构并与文献中的生化数据一致，提出了核酶作用的催化机制。抗HIV - 1 gag核酶与其流产DNA底物之间的复合物在检测到连续的A.T碱基对轨迹时得以体现；这表明核酶与其DNA底物之间的相互作用比在游离核酶情况下观察到的更强，在游离核酶中，茎I和茎III区域的碱基与核酶核心区域有强烈相互作用（萨尔马，R. H.等人，《欧洲生物化学学会联合会快报》375，317 - 23，1995）。复合物的形成在设计合适的治疗性核酶方面提供了一定的指导方针。如果核酶茎区域的残基与保守核心相互作用，它可能会阻止或干扰催化活性三级结构的形成。
