Chowrira B M, Berzal-Herranz A, Burke J M
Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington 05405.
Biochemistry. 1993 Feb 2;32(4):1088-95. doi: 10.1021/bi00055a014.
Metal ion requirements for RNA binding, cleavage, and ligation by the hairpin ribozyme have been analyzed. RNA cleavage is observed when Mg2+, Sr2+, or Ca2+ are added to a 40 mM Tris-HCl buffer, indicating that these divalent cations were capable of supporting the reaction. No reaction was observed when other ions (Mn2+, Co2+, Cd2+, Ni2+, Ba2+, Na+, K+, Li+, NH4+, Rb+, and Cs+) were tested. In the absence of added metal ions, spermidine can induce a very slow ribozyme-catalyzed cleavage reaction that is not quenched by chelating agents (EDTA and EGTA) that are capable of quenching the metal-dependent reaction. Addition of Mn2+ to a reaction containing 2 mM spermidine increases the rate of the catalytic step by at least 100-fold. Spermidine also reduces the magnesium requirement for the reaction and strongly stimulates activity at limiting Mg2+ concentrations. There are no special ionic requirements for formation of the initial ribozyme-substrate complex--analysis of complex formation using native gels and kinetic assays shows that the ribozyme can bind substrate in 40 mM Tris-HCl buffer. Complex formation is inhibited by both Mn2+ and Co2+. Ionic requirements for the ribozyme-catalyzed ligation reaction are very similar to those for the cleavage reaction. We propose a model for catalysis by the hairpin ribozyme that is consistent with these findings. Formation of an initial ribozyme-substrate complex occurs without the obligatory involvement of divalent cations. Ions (e.g., Mg2+) can then bind to form a catalytically proficient complex, which reacts and dissociates.(ABSTRACT TRUNCATED AT 250 WORDS)
对发夹状核酶进行RNA结合、切割和连接所需的金属离子进行了分析。当向40 mM Tris-HCl缓冲液中添加Mg2+、Sr2+或Ca2+时，可观察到RNA切割，这表明这些二价阳离子能够支持该反应。当测试其他离子（Mn2+、Co2+、Cd2+、Ni2+、Ba2+、Na+、K+、Li+、NH4+、Rb+和Cs+）时，未观察到反应。在没有添加金属离子的情况下，亚精胺可诱导非常缓慢的核酶催化切割反应，该反应不会被能够淬灭金属依赖性反应的螯合剂（EDTA和EGTA）淬灭。向含有2 mM亚精胺的反应中添加Mn2+可使催化步骤的速率提高至少100倍。亚精胺还降低了反应对镁的需求，并在有限的Mg2+浓度下强烈刺激活性。形成初始核酶-底物复合物没有特殊的离子要求——使用天然凝胶和动力学分析对复合物形成的分析表明，核酶可以在40 mM Tris-HCl缓冲液中结合底物。Mn2+和Co2+均抑制复合物的形成。核酶催化连接反应的离子要求与切割反应非常相似。我们提出了一个与这些发现一致的发夹状核酶催化模型。初始核酶-底物复合物的形成无需二价阳离子的必然参与。然后离子（如Mg2+）可以结合形成催化活性复合物，该复合物发生反应并解离。（摘要截于250字）
