The kinetic mechanism of the hairpin ribozyme in vivo:: Influence of RNA helix stability on intracellular cleavage kinetics
The kinetic mechanism of the hairpin ribozyme in vivo:: Influence of RNA helix stability on intracellular cleavage kinetics
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DOI：
10.1006/jmbi.1999.3380
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发表时间：
2000-01-21
影响因子：
5.6
通讯作者：
Fedor, MJ
中科院分区：
文献类型：
--
作者：
Donahue, CP;Yadava, RS;Fedor, MJ
发夹核酶结构、裂解和连接动力学以及平衡之间的关系已经在各种体外反应条件下得到了广泛的表征。我们开发了酵母发夹核酶切割活性的定量分析，以了解体外RNA酶的结构-功能关系如何与细胞中RNA介导的反应相关。在这里，我们报告了一个重要的二级结构元素H1的稳定性变化对细胞内切割动力学的影响。H1是在核酶和3'切割产物rna之间形成的碱基配对螺旋。少于3个碱基对的H1序列在体外或体内都不能支持充分的活性，这表明短RNA螺旋在体外和细胞内条件下的稳定性没有任何显著差异。在含有10 mM MgCl2的标准体外条件下，核酶结合产物的切割和连接之间的内部平衡有利于连接。因此，具有稳定H1序列的核酶表现出急剧降低的自裂解率，因为裂解被结合产物的快速重新连接所逆转。相比之下，在H1中有多达26个碱基对的核酶在体内继续以最大速率自裂。如果细胞内条件促进了产物的快速解离或改变了内部平衡以有利于裂解，则可以解释大产物等抑制裂解的失败。体外模型实验表明，在细胞内离子条件下，犬产品的分裂和结扎之间的内部平衡可能有利于分裂。(C) 2000年学术出版社。
The relationship between hairpin ribozyme structure, and cleavage and ligation kinetics, and equilibria has been characterized extensively under a variety of reaction conditions in vitro. We developed a quantitative assay of hairpin ribozyme cleavage activity in yeast to learn how structure-function relationships defined for RNA enzymes in vitro relate to RNA-mediated reactions in cells. Here, we report the effects of variation in the stability of an essential secondary structure element, H1, on intracellular cleavage kinetics. H1 is the base-paired helix formed between ribozyme and 3' cleavage product RNAs. H1 sequences with fewer than three base-pairs fail to support full activity in vitro or in vivo, arguing against any significant difference in the stability of short RNA helices under in vitro and intracellular conditions. Under standard conditions in vitro that include 10 mM MgCl2, the internal equilibrium between cleavage and ligation of ribozyme-bound products favors ligation. Consequently, ribozymes with stable H1 sequences display sharply reduced self-cleavage rates, because cleavage is reversed by rapid re-ligation of bound products. In contrast, ribozymes with as many as 26 base-pairs in H1 continue to self-cleave at maximum rates in vivo. The failure of large products tc, inhibit cleavage could be explained if intracellular conditions promote rapid product dissociation or shift the internal equilibrium to favor cleavage. Model experiments in vitro suggest that the internal equilibrium between cleavage and ligation of hound products is likely to favor cleavage under intracellular ionic conditions. (C) 2000 Academic Press.