An improved version of the hairpin ribozyme functions as a ribonucleoprotein complex.
An improved version of the hairpin ribozyme functions as a ribonucleoprotein complex.
复制标题
发夹核酶的改进版本起到核糖核蛋白复合物的作用。
DOI：
10.1021/bi00023a021
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Burke,JM
中科院分区：
文献类型：
--
作者：
Sargueil,B;Pecchia,DB;Burke,JM
1995年3月31日接收的摘要：大多数具有催化活性的RNA分子在细胞内以核糖核蛋白的形式发挥作用。这些复合物通常很大，定义不清，难以研究。作为研究核糖核蛋白生物催化的模型系统，我们已经通过插入RNA结构来修饰发夹状核酶，该RNA结构作为噬菌体R17外壳蛋白的结合位点，以核酶螺旋4的延伸形式，该延伸位于催化结构域的外围。在不存在蛋白质的情况下，我们发现蛋白质结合结构域的掺入使发夹核酶的催化效率增加2倍用于切割反应和16倍用于连接反应。这种活性的增加与折叠成活性三级结构的分子比例的增加相关，如通过UV交联测定所测量的。复合物形成的迁移率和过滤器结合试验表明，R17外壳蛋白bindsto的嵌合核酶的解离常数基本上相同的分离的蛋白质结合结构域的，没有结合的蛋白质的未修饰的核酶可以检测到。切割和连接反应的动力学不改变的存在下饱和浓度的外壳蛋白，竞争研究表明，蛋白质仍然boundto核酶在整个催化循环。这些研究证实发夹状核酶可以在体外以核糖核蛋白的形式有效地发挥功能，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结构域，例如HIV-1 Rev-结合和tar元件，其可能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。核酶是通过体外研究显示在没有蛋白质的情况下催化反应的RNA分子。虽然许多不同的催化RNA基序已被表征，但已知许多生物反应不是由RNA单独介导，而是由核糖核蛋白（RNP）1复合物介导。RNP介导的反应位于中央细胞途径的核心，包括剪接体催化的核前mRNA剪接、核糖体蛋白合成和信号识别颗粒（SRP）介导的蛋白跨膜转运（综述见Wittop Koning和Schiimperli（1994））。即使在RNA分子在体外没有蛋白质的情况下发挥作用，也有强有力的遗传和生物化学证据表明蛋白质因子对体内催化功能至关重要。两个值得注意的例子是核糖核酸酶P，其中蛋白辅因子（大肠杆菌中的C5蛋白）在体内是需要的，但在体外在高离子强度下不需要（Guerrier-Takada et al.，1988;赖希等人，1988年，A
Revised Manuscript Received March 31, 1995® abstract: 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 bindsto 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 boundto 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 influencingsubcellular localization, regulating intracellular activity, or generating ribozymes that also function as “decoys” in antiviral applications.Ribozymes are RNA molecules that have been shown, through in vitro studies, to catalyze reactions in the absence of proteins. Although a number of different catalytic RNA motifs have been characterized, many biological reactions are known to be mediated not by RNA alone but by ribonucleoprotein (RNP) 1 complexes. RNP-mediated reac-tions lie at the heart of central cellular pathways, including nuclear pre-mRNA splicing catalyzed by the spliceosome, ribosomal protein synthesis, and protein translocation across membranes mediated by the signal recognition particle (SRP)(for review, see Wittop Koning and Schiimperli (1994)). Even in the case of RNA molecules that function without proteins in vitro, there is strong genetic and biochemical evidence that protein factors are essential for catalytic function in vivo. Two noteworthy examples are ribonuclease P, where a protein cofactor (C5 protein in Escherichia coli) is required in vivo but notat high ionic strength in vitro (Guerrier-Takada et al., 1988; Reich et al., 1988), and a