Chowrira B M, Berzal-Herranz A, Keller C F, Burke J M
Markey Center for Molecular Genetics, Department of Microbiology and Molecular Genetics, University of Vermont, Burlington 05405.
J Biol Chem. 1993 Sep 15;268(26):19458-62.
The hairpin ribozyme catalyzes site-specific cleavage of an RNA substrate using a magnesium-dependent transphosphorylation mechanism. Here, we describe experiments designed to test the importance of ribose 2'-hydroxyl groups for ribozyme function. Ribozymes for this work were synthesized in two segments using solid-phase RNA phosphoramidite chemistry. 2'-Deoxyribonucleotides were systematically introduced at each of the 50 positions within the ribozyme, and the catalytic activity of the resulting mixed RNA-DNA polymers was measured. Deletion of the 2'-hydroxyl group at each of four sites (A10, G11, A24, and C25) was found to result in severe inhibition of cleavage activity (kcat/KM decreased by 100- to 1000-fold), although KM measurements and mobility-shift assays showed that substrate binding was not affected. Identical results were obtained upon substitution of these ribonucleotides with 2'-O-methyl derivatives. Inhibition by 2'-modified sugars at G11 or A24 was rescued by increased Mg2+ concentrations, suggesting that these 2'-hydroxyls may function in magnesium binding. Our results demonstrate that the 2'-hydroxyl groups at A10, G11, A24, and C25 provide essential functions for catalysis, possibly forming important tertiary contacts or magnesium coordination sites that are necessary for active site architecture.
发夹状核酶利用镁依赖性转磷酸化机制催化RNA底物的位点特异性切割。在此，我们描述了旨在测试核糖2'-羟基基团对核酶功能重要性的实验。用于这项工作的核酶是使用固相RNA亚磷酰胺化学方法分两段合成的。在核酶内的50个位置中的每一个位置系统地引入2'-脱氧核糖核苷酸，并测量所得混合RNA-DNA聚合物的催化活性。发现四个位点（A10、G11、A24和C25）中的每一个位点的2'-羟基缺失都会导致切割活性受到严重抑制（kcat/KM降低100至1000倍），尽管KM测量和迁移率变动分析表明底物结合不受影响。用2'-O-甲基衍生物取代这些核糖核苷酸后获得了相同的结果。G11或A24处的2'-修饰糖的抑制作用可通过增加Mg2+浓度来挽救，这表明这些2'-羟基可能在镁结合中起作用。我们的结果表明，A10、G11、A24和C25处的2'-羟基基团为催化提供了基本功能，可能形成了活性位点结构所需的重要三级接触或镁配位位点。
