Phosphorothioate substitution can substantially alter RNA conformation.
Phosphorothioate substitution can substantially alter RNA conformation.
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DOI：
10.1021/bi992712b
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发表时间：
2000-04
期刊：
影响因子：
2.9
通讯作者：
jeff smith;E. Nikonowicz
中科院分区：
文献类型：
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作者：
jeff smith;E. Nikonowicz
硫代磷酸酯取代干扰实验，通常用于立体特异性地确定参与RNA-配体结合和RNA-定向催化的磷酰氧位点，其解释基于未经测试的假设，即取代不会改变修饰分子的构象，使其脱离生物活性状态。使用NMR光谱，我们已经测试了这一假设，通过确定立体特异性硫代磷酸酯取代在5个位置的RNA发夹中含有噬菌体MS 2衣壳蛋白的结合位点的结构效应。在大多数位点，取代几乎没有影响，导致磷酸骨架的微小扰动并增加发夹环中核苷酸之间的堆积。然而，在一个位点，硫代磷酸取代导致形成衣壳蛋白-RNA复合物所必需的未配对腺嘌呤从RNA螺旋环出进入大沟。这些结果表明，硫代磷酸酯取代可以大大改变RNA的构象在不规则的二级结构的位置，复杂的使用取代干扰实验来研究RNA的结构和功能。
Phosphorothioate substitution-interference experiments, routinely used to stereospecifically identify phosphoryl oxygen sites that participate in RNA-ligand binding and RNA-directed catalysis, rest in their interpretation on the untested assumption that substitution does not alter the conformation of the modified molecule from its biologically active state. Using NMR spectroscopy, we have tested this assumption by determining the structural effect of stereospecific phosphorothioate substitution at five positions in an RNA hairpin containing the binding site for bacteriophage MS2 capsid protein. At most sites, substitution has little or no effect, causing minor perturbations in the phosphate backbone and increasing the stacking among nucleotides in the hairpin loop. At one site, however, phosphorothioate substitution causes an unpaired adenine necessary for formation of the capsid protein-RNA complex to loop out of the RNA helix into the major groove. These results indicate that phosphorothioate substitution can substantially alter the conformation of RNA at positions of irregular secondary structure, complicating the use of substitution-interference experiments to study RNA structure and function.