Rational design of allosteric ribozymes
Rational design of allosteric ribozymes
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DOI：
10.1016/s1074-5521(97)90197-6
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发表时间：
1997-06-01
影响因子：
--
通讯作者：
Breaker, RR
中科院分区：
文献类型：
--
作者：
Tang, J;Breaker, RR
背景：细胞过程的有效运行依赖于每个细胞对其代谢途径施加的严格控制。一些蛋白质酶受变构调节，位于酶活性部位之外的结合部位可以特异性地识别效应分子，并通过构象变化改变酶的催化速率。虽然RNA也进行化学反应，但在生物系统中，没有核酶作为真正的变构酶起作用。最近已经证实，小分子受体可以很容易地由RNA组成，正如体外选择的各种能与相应配体分子特异结合的RNA适配子所证明的那样。我们通过设计连接的适体-核酶复合体来检测现有核酶的催化活性是否可以控制在效应分子的控制之下。结果：通过将ATP结合的RNA连接到自切割的核酶，我们创造了第一个变构核酶的催化速度可以由ATP控制的例子。当添加腺苷或三磷酸腺苷时，反应速率降低180倍，但在d三磷酸腺苷或其他核苷三磷酸盐的存在下，没有检测到抑制作用。适体结构域中的突变有望消除ATP结合或增加适体和核酶结构域之间的距离，导致ATP特异性变构控制的丧失。使用类似的设计方法，我们创建了在ATP存在下被激活的变构锤头状核酶，并创建了另一种可由茶碱控制的核酶。结论：这些连接的适体-核酶的催化特性表明，催化RNA也可以受到变构调节--这是某些蛋白质酶的一个关键特征。此外，通过使用简单合理的设计策略，现在有可能设计出新的催化多核苷酸，其速率可以由小效应器分子紧密和特定地控制。
Background: Efficient operation of cellular processes relies on the strict control that each cell exerts over its metabolic pathways. Some protein enzymes are subject to allosteric regulation, in which binding sites located apart from the enzyme's active site can specifically recognize effector molecules and alter the catalytic rate of the enzyme via conformational changes. Although RNA also performs chemical reactions, no ribozymes are known to operate as true allosteric enzymes in biological systems. It has recently been established that small-molecule receptors can readily be made of RNA, as demonstrated by the in vitro selection of various RNA aptamers that can specifically bind corresponding ligand molecules. We set out to examine whether the catalytic activity of an existing ribozyme could be brought under the control of an effector molecule by designing conjoined aptamer-ribozyme complexes.Results: By joining an ATP-binding RNA to a self-cleaving ribozyme, we have created the first example of an allosteric ribozyme that has a catalytic rate that can be controlled by ATP. A 180-fold reduction in rate is observed upon addition of either adenosine or ATP, but no inhibition is detected in the presence of dATP or other nucleoside triphosphates. Mutations in the aptamer domain that are expected to eliminate ATP binding or that increase the distance between aptamer and ribozyme domains result in a loss of ATP-specific allosteric control. Using a similar design approach, allosteric hammerhead ribozymes that are activated in the presence of ATP were created and another ribozyme that can be controlled by theophylline was created.Conclusions: The catalytic features of these conjoined aptamer-ribozyme constructs demonstrate that catalytic RNAs can also be subject to allosteric regulation - a key feature of certain protein enzymes. Moreover, by using simple rational design strategies, it is now possible to engineer new catalytic polynucleotides which have rates that can be tightly and specifically controlled by small effector molecules.