The internal equilibrium of the hairpin ribozyme: Temperature, ion and pH effects
The internal equilibrium of the hairpin ribozyme: Temperature, ion and pH effects
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DOI：
10.1006/jmbi.1999.2543
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发表时间：
1999-03-05
影响因子：
5.6
通讯作者：
Fedor, MJ
中科院分区：
文献类型：
--
作者：
Nesbitt, SM;Erlacher, HA;Fedor, MJ
发夹核酶可逆地切割RNA底物的磷酸二酯以产生具有5'羟基和2'，3 '-环状磷酸末端的产物。我们先前发现，在标准条件下，连接的速率常数比切割的速率常数快十倍。锤头状核酶催化相同的反应，但据报道，在相同条件下有利于切割相对于连接超过100倍。为了探索这种差异的基础，我们研究了温度，离子和pH值对发夹状核酶内部平衡的影响。在相同的条件下，与连接相关的熵的损失是发夹比锤头状核酶少，这与以下概念相一致，即更刚性的发夹结构在连接时比更柔性的锤头状结构经历更小的动力学降低。增加的盐和降低的温度使平衡向连接方向移动，而pH几乎没有影响，这表明稳定RNA结构的条件倾向于促进连接。发夹核酶似乎在连接时吸收至少一个三价或二价阳离子或两个单价阳离子。不同阳离子促进连接的效率强烈地依赖于化合价，而不太强烈地依赖于离子半径或电负性。阳离子选择性的这种模式表明，阳离子通过离域静电屏蔽促进连接，可能与连接的核酶中特别高的电荷密度的区域相互作用。离子条件的变化产生大的但补偿的焓和熵的变化，用于裂解和连接。因此，除了与切割相关的核酶动力学的任何增加之外，相关阳离子的重组显著有助于发夹核酶热力学。(C)北京：科学出版社.
The hairpin ribozyme reversibly cleaves phosphodiesters of RNA substrates to generate products with 5' hydroxyl and 2',3'-cyclic phosphate termini. We previously found that the rate constant for Ligation is tenfold faster than the rate constant for cleavage under standard conditions. The hammerhead ribozyme catalyzes the same reactions but is reported to favor cleavage relative to ligation by more than 100-fold under the same conditions. To explore the basis for this difference, we examined the influence of temperature, ions and pH on the hairpin ribozyme internal equilibrium. Under the same conditions, the loss of entropy associated with ligation is less for the hairpin than for the hammerhead ribozyme, consistent with the notion that a more rigid hairpin structure undergoes a smaller decrease in dynamics upon ligation than the more flexible hammerhead structure. Increased salt and reduced temperature shift the equilibrium toward ligation while pH has little effect, suggesting that conditions that stabilize RNA structure tend to promote Ligation. The hairpin ribozyme appears to take up at least one tri- or divalent cation or two monovalent cations upon ligation. The efficiency with which different cations promote ligation depends strongly on valence and, less strongly, on ionic radius or electronegativity. This pattern of cation selectivity suggests that cations promote ligation through delocalized electrostatic shielding, perhaps interacting with a region of especially high charge density in the ligated ribozyme. Changes in ionic conditions produce large but compensating changes in enthalpy and entropy for cleavage and ligation. Thus, in addition to any increase in ribozyme dynamics associated with cleavage, re-organization of associated cations contributes significantly to hairpin ribozyme thermodynamics. (C) 1999 Academic Press.