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首页> 外文期刊>Biochemistry >AN IMPROVED VERSION OF THE HAIRPIN RIBOZYME FUNCTIONS AS A RIBONUCLEOPROTEIN COMPLEX
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AN IMPROVED VERSION OF THE HAIRPIN RIBOZYME FUNCTIONS AS A RIBONUCLEOPROTEIN COMPLEX

机译：作为一种核糖蛋白复合物的发夹核酶功能的改进版本

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摘要

Most RNA molecules that are endowed with catalytic activity function in the form of ribonucleoproteins within cells. These complexes are frequently large, poorly defined, and difficult to study. As a model system to study biological catalysis by ribonucleoproteins, we have modified the hairpin ribozyme by inserting an RNA structure that serves as a binding site for bacteriophage R17 coat protein in the form of an extension to ribozyme helix 4, which lies at the periphery of the catalytic domain. In the absence of protein, we find that incorporation of the protein-binding domain increases the catalytic efficiency of the hairpin ribozyme by 2-fold for the cleavage reaction and 16-fold for the ligation reaction. This increase in activity correlates with an increase in the proportion of molecules which fold into the active tertiary structure, as measured by a UV cross-linking assay. Mobility-shift and filter-binding assays of complex formation show that R17 coat protein binds to the chimeric ribozyme with a dissociation constant essentially identical to that of the isolated protein-binding domain; no binding of the protein to the unmodified ribozyme could be detected. The kinetics of cleavage and ligation reactions are not altered by the presence of saturating concentrations of coat protein, and competition studies demonstrate that the protein remains bound to the ribozyme throughout the catalytic cycle. These studies establish that the hairpin ribozyme can be engineered to function efficiently in the form of a ribonucleoprotein in vitro and will serve as the basis for future experimentation to understand mechanisms of protein modulation of catalytic RNA activity, and to introduce other protein-binding domains, for example, HIV-1 rev-binding and tar elements, which may be useful for influencing subcellular localization, regulating intracellular activity, or generating ribozymes that also function as ''decoys'' in antiviral applications.
机译：大多数具有催化活性的RNA分子以核糖核蛋白的形式在细胞内发挥作用。这些复合物通常很大，定义不清且难以研究。作为研究核糖核蛋白生物催化的模型系统，我们通过插入一个RNA结构来修饰发夹状核酶，该结构作为噬菌体R17外壳蛋白的结合位点以核糖酶螺旋4的延伸形式存在，位于核糖核酸螺旋的外围。催化域。在不存在蛋白质的情况下，我们发现掺入蛋白质结合结构域可使发夹状核酶的催化效率提高2倍（裂解反应）和16倍（连接反应）。活性的这种增加与折叠成活性三级结构的分子的比例的增加有关，如通过UV交联测定法所测量的。复合物形成的迁移率漂移和滤膜结合试验表明，R17外壳蛋白与嵌合核酶结合的解离常数与分离的蛋白质结合域的解离常数基本相同。没有检测到蛋白质与未修饰的核酶的结合。裂解和连接反应的动力学不会因存在饱和浓度的外壳蛋白而改变，竞争研究表明该蛋白在整个催化循环中仍与核酶结合。这些研究表明，发夹状核酶可以进行工程改造以在体外以核糖核蛋白的形式有效发挥功能，并将作为未来实验的基础，以了解催化RNA活性的蛋白质调节机制并引入其他蛋白质结合域，例如，HIV-1 rev-binding和tar元素，可用于影响亚细胞定位，调节细胞内活性或产生在抗病毒应用中也起“诱饵”作用的核酶。

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