Effects of background anionic compounds on the activity of the hammerhead ribozyme in Mg2+-unsaturated solutions
Effects of background anionic compounds on the activity of the hammerhead ribozyme in Mg2+-unsaturated solutions
复制标题
Mg2-不饱和溶液中背景阴离子化合物对锤头核酶活性的影响
DOI：
10.1007/s00775-015-1286-y
复制
发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Naoki Sugimoto
中科院分区：
文献类型：
--
作者：
Shu-ichi Nakano;Yuichi Kitagawa;Daisuke Miyoshi;Naoki Sugimoto
细胞核酶在含有大量阴离子化合物和大分子的介质中表现出催化活性。在这项研究中，锤头状核酶的RNA切割活性的Mg ~（2+）诱导的使用含有背景核酸，小磷酸盐和羧酸化合物，和中性聚合物的溶液进行了研究。底物裂解动力学分析表明，阴离子化合物不影响核酶的活性在Mg 2 +-饱和溶液中，几乎没有影响的阴离子-Mg 2+复合物形成。另一方面，在Mg 2 +-不饱和溶液中底物裂解的速率在细胞中发现的阴离子化合物的高背景条件下降低。减少的程度是更多的具有更大的净负电荷，所造成的减少量的Mg 2+，可用于核酶反应。值得注意的是，当比较相同数量的磷酸基团的影响时，具有磷酸二酯键的背景DNA和RNA分子与具有-2电荷的腺苷单磷酸一样降低切割速率。在形成胶束的脂肪酸的背景中也观察到比从分子电荷预期的速率更大的降低。此外，聚（乙二醇）的解决方案部分恢复了核酶的活性，这表明可能的作用，大分子拥挤在抵消背景阴离子对核酶反应的抑制作用。这些结果具有生物学和实际意义方面的影响，分子环境的离子结合到RNA的效率。
Cellular ribozymes exhibit catalytic activity in media containing large numbers of anionic compounds and macromolecules. In this study, the RNA cleavage activity of the hammerhead ribozyme induced by Mg2+was investigated using the solutions containing background nucleic acids, small phosphate and carboxylic acid compounds, and neutral polymers. Analysis of the substrate cleavage kinetics showed that the anionic compounds do not affect the ribozyme activity in Mg2+-saturated solutions and there is almost no effect of the anion–Mg2+complexes formed. On the other hand, the rate of substrate cleavage in Mg2+-unsaturated solutions was reduced under conditions of a high background of anionic compounds found in cells. The extent of the reduction was more with a greater net negative charge, caused by decreased amounts of Mg2+that could be used for the ribozyme reaction. It was remarkable that background DNA and RNA molecules having phosphodiester bonds reduced the cleavage rate as much as adenosine monophosphates having a charge of −2 when the effects of the same amount of phosphate groups were compared. Greater reductions in rates than those expected from the molecular charge were also observed in the background of fatty acids that form micelles. An addition of poly(ethylene glycol) to the solutions partially restored the ribozyme activity, suggesting a possible role of macromolecular crowding in counteracting the inhibitory effects of background anions on the ribozyme reaction. The results have biological and practical implications with respect to the effects of molecular environment on the efficiency of ion binding to RNA.