Characterization of a native hammerhead ribozyme derived from schistosomes
Characterization of a native hammerhead ribozyme derived from schistosomes
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DOI：
10.1261/rna.7950605
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发表时间：
2005-02-01
期刊：
影响因子：
4.5
通讯作者：
DeRose, VJ
中科院分区：
文献类型：
--
作者：
Osborne, EM;Schaak, JE;DeRose, VJ
最近重新审查的作用的螺旋周围的锤头状核酶的保守的核心已经确定了推定的环-环之间的相互作用茎I和II在本地锤头状序列。这些延伸的锤头序列在低浓度的二价阳离子下比最小锤头更有活性。环-环相互作用被提出来稳定保守核心的更活跃的构象。在这里，一个扩展的锤头序列来自曼氏血吸虫的动力学和热力学特征进行。观察到两相动力学，表明存在至少两种构象，一种裂解速度快，另一种裂解速度慢。用设计用于消除两个环之间的相互作用的聚（U）序列替换环II导致活性大大降低，表明环-环相互作用确实有助于形成更活性的构象。先前使用最小锤头的研究已经显示了在切割位点和A9的5 '处的R-p-硫代磷酸酯取代的有害影响，这两者都可以用Cd 2+拯救。在此，在切割位点处和A9的5 '处的硫代磷酸酯修饰在核糖体衍生的序列中进行。在Mg2+，两个硫代磷酸酯取代减少了整体馏分切割没有显着影响所观察到的裂解率。在这两种情况下，添加Cd2+拯救切割，这表明这些仍然是假定的金属结合位点在这个天然序列。
A recent re-examination of the role of the helices surrounding the conserved core of the hammerhead ribozyme has identified putative loop-loop interactions between stems I and II in native hammerhead sequences. These extended hammerhead sequences are more active at low concentrations of divalent cations than are minimal hammerheads. The loop-loop interactions are proposed to stabilize a more active conformation of the conserved core. Here, a kinetic and thermodynamic characterization of an extended hammerhead sequence derived from Schistosoma mansoni is performed. Biphasic kinetics are observed, suggesting the presence of at least two conformers, one cleaving with a fast rate and the other with a slow rate. Replacing loop II with a poly(U) sequence designed to eliminate the interaction between the two loops results in greatly diminished activity, suggesting that the loop-loop interactions do aid in forming a more active conformation. Previous studies with minimal hammerheads have shown deleterious effects of R-p-phosphorothioate substitutions at the cleavage site and 5' to A9, both of which could be rescued with Cd2+. Here, phosphorothioate modifications at the cleavage site and 5' to A9 were made in the schistosome-derived sequence. In Mg2+, both phosphorothioate substitutions decreased the overall fraction cleaved without significantly affecting the observed rate of cleavage. The addition of Cd2+ rescued cleavage in both cases, suggesting that these are still putative metal binding sites in this native sequence.