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Functional structures of nucleic acids and their recognition by proteins

核酸的功能结构及其蛋白质的识别

基本信息

批准号：
07458146
负责人：
SUGIMOTO Naoki
金额：
$ 3.84万
依托单位：
KONAN UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07458146/
关键词：
Ribozyme Gene cleavage Loop structure Lead ion Rare earth ion Nearest-neighbor model Gel electrophoresis Reaction mechanism RecA蛋白質オリゴペプチド DNA結合蛋白質表面プラズモン共鳴円二色性核酸反応速度構造安定性

项目摘要

A ribozyme in an RNA enzyme that can catalyze a biochemical reaction. Because a ribozyme requires a metal ions for a catalytic function, the ribozyme is considered as one of the metalloenzymes. Ribozyme reactions are very sensitive to species and concentration of metal ions, suggesting that divalent metal ions may play important roles for the chemical reaction and structural stabilization of the ribozyme.We have investigated the combined effects of metal ions on both the structural stabilization of the complex between a ribozyme (a leadzyme), CUGGGAGUCC,and a substrate, GGACCGAGCCAG,and the cleavage step in the active center of the complex has been also investigated kinetically.The results showed that the addition of Nd^<3+> in the presence of Pb^<2+> increased significantly the yield of the RNA cleavage reaction by the leadzyme, although other rare earth ions or divalent ions except Pb^<2+> did not promote the reaction. Further, kinetics for the leadzyme reaction was measured at various concentrations of Nd^<3+> and Pb^<2+>. At low concentration rations of Nd^<3+> under a constant total concentration of metal ions, Nd^<3+> increased the stablility of the complex between the leadzyme and the substrate. In contrast, at high concentration ratios of Nd^<3+>, the addition of Nd^<3+> decreased the stability of the complex. The rate constant of the cleavage step was maximized when the ratio of Nd^<3+> to Pb^<2+> was 1 : 1. These results suggest that the complex between the leadzyme and the substrate has binding sites for Nd^<3+> ion taht influence complex stability and catalyze directly the cleavage reaction. On the basis of the results, we propose a two-metal-ion mechanism in which Pb^<2+> and Nd^<3+> play the roles of base and acid catalyst, respectively.

RNA酶中的核酶，可以催化生化反应。由于核酶需要金属离子来发挥催化功能，因此核酶被认为是金属酶之一。核酶反应对金属离子的种类和浓度非常敏感，这表明二价金属离子可能对核酶的化学反应和结构稳定发挥重要作用。我们研究了金属离子对核酶（先导酶）CUGGGAGUCC和底物GGACCGAGCCAG之间复合物结构稳定性的综合影响，以及核酶中的裂解步骤结果表明，在Pb^<2+>存在的情况下，添加Nd^<3+>显着提高了铅酶RNA裂解反应的产率，但除Pb^<2+>之外的其他稀土离子或二价离子并不促进该反应。此外，在不同浓度的Nd 3+ 和Pb 2+ 下测量了引导酶反应的动力学。在金属离子总浓度恒定的低Nd 3+ 浓度比下，Nd 3+ 增加了引导酶和底物之间的复合物的稳定性。相反，在高浓度比的Nd ^ 3+ 下，Nd ^ 3+ 的添加降低了络合物的稳定性。当 Nd^<3+> 与 Pb^<2+> 的比率为 1:1 时，裂解步骤的速率常数最大化。这些结果表明，引导酶和底物之间的复合物具有 Nd^<3+> 离子的结合位点，这会影响复合物的稳定性并直接催化裂解反应。基于这些结果，我们提出了一种双金属离子机理，其中Pb^<2+>和Nd^<3+>分别起碱催化剂和酸催化剂的作用。