Pyle A M, McSwiggen J A, Cech T R
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Proc Natl Acad Sci U S A. 1990 Nov;87(21):8187-91. doi: 10.1073/pnas.87.21.8187.
Like protein enzymes, RNA enzymes (ribozymes) provide specific binding sites for their substrates. We now show that equilibrium dissociation constants for complexes between the Tetrahymena ribozyme and its RNA substrates and products can be directly measured by electrophoresis in polyacrylamide gels containing divalent cations. Binding is 10(3)- to 10(4)-fold tighter (4-5 kcal/mol at 42 degrees C) than expected from base-pairing interactions alone, implying that tertiary interactions also contribute to energetic stabilization. Binding decreases with single base changes in the substrate, substitution of deoxyribose sugars, and lower Mg2+ concentration. Ca2+, which enables the ribozyme to fold but is unable to mediate efficient RNA cleavage, promotes weaker substrate binding than Mg2+. This indicates that Mg2+ has special roles in both substrate binding and catalysis. Mutagenesis of a region near the internal guide sequence disrupts substrate binding, whereas binding is not significantly affected by a mutation of the guanosine-binding site. This approach should be generally useful for analysis of ribozyme variants independent of their catalytic activities.
与蛋白质酶一样，RNA酶（核酶）为其底物提供特定的结合位点。我们现在表明，通过在含有二价阳离子的聚丙烯酰胺凝胶中进行电泳，可以直接测量四膜虫核酶与其RNA底物和产物之间复合物的平衡解离常数。结合比仅基于碱基配对相互作用所预期的紧密10³至10⁴倍（42℃时为4 - 5千卡/摩尔），这意味着三级相互作用也有助于能量稳定。底物中的单碱基变化、脱氧核糖的取代以及较低的Mg²⁺浓度都会导致结合减弱。Ca²⁺能使核酶折叠，但无法介导有效的RNA切割，与Mg²⁺相比，它促进的底物结合较弱。这表明Mg²⁺在底物结合和催化中都具有特殊作用。内部引导序列附近区域的诱变会破坏底物结合，而鸟苷结合位点的突变对结合没有显著影响。这种方法对于分析核酶变体应普遍有用，而与它们的催化活性无关。
