KINETICS AND THERMODYNAMICS OF TRIPLE-HELIX FORMATION - EFFECTS OF IONIC-STRENGTH AND MISMATCHES
KINETICS AND THERMODYNAMICS OF TRIPLE-HELIX FORMATION - EFFECTS OF IONIC-STRENGTH AND MISMATCHES
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DOI：
10.1021/bi00153a021
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发表时间：
1992-09-29
期刊：
影响因子：
2.9
通讯作者：
HELENE, C
中科院分区：
文献类型：
--
作者：
ROUGEE, M;FAUCON, B;HELENE, C
从热变性和复性UV吸光度曲线确定高嘌呤-高嘧啶22-碱基对双链体（嘌呤链序列：5'd [AAAGGAGGAGAAGAAAAA] 3 '）和4条22-dN第三链（22 dN：5'd [TTTCCTCCTCTNCTTCTTTTTT] 3'，其中N = A、C、T或G）之间三链体形成反应的热力学和动力学参数。冷却和加热曲线是不可重叠的，因此，我们可以确定作为温度的函数的缔合（k（on））和解离（k（off））的速率常数，假设一个类似于为形成反应开发的双态模型。实验在10 mM二甲胂酸盐缓冲液（pH 6.8）中在NaCl浓度范围为20至300 mM的情况下进行。在实验精度内，主要结果如下：（i）速率常数k（on）和k（off）导致线性Arrhenius图，与双态缔合和解离的预测一致。(ii)k（on）与位于第三链中心的碱基N的性质无关。(iii)当NaCl浓度降低时，k（on）强烈降低。(iv)活化能E（on）始终为负值，并且随着NaCl浓度的降低而变得更负。(v)k（off）与NaCl浓度无关，但取决于碱N，其大小遵循C > G > A >> T的顺序。(vi)活化能E（off）与基数N无关。所有这些结果都是根据一个成核拉链模型进行讨论的，该模型类似于为双螺旋转变而开发的模型[克雷格，M。E、Crothers，D. M.，& Doty，P.（1971）J. Mol. 62，383 -401; Porschke，D.，艾根，M。等（1971）J. Mol. 62，361-381]。
Thermodynamic and kinetic parameters for the triplex-forming reactions between a homopurine-homopyrimidine 22-base-pair duplex (sequence of the purine strand: 5'd[AAAGGAGGAGAAGAAGAAAAAA]3') and the four 22-dN third strands (22 dN: 5'd[TTTCCTCCTCTNCTTCTTTTTT]3', where N = A, C, T, or G) were determined from thermal denaturation and renaturation UV absorbance profiles. Cooling and heating curves were not superimposable and thus allowed us to determine the rate constants of association (k(on)) and dissociation (k(off)) as a function of temperature, assuming a two-state model analogous to that developed for duplex-forming reactions. Experiments were performed in 10 mM cacodylate buffer (pH 6.8) in the presence of NaCl concentrations ranging from 20 to 300 mM. Within experimental accuracy, the main results are the following: (i) The rate constants k(on) and k(off) result in linear Arrhenius plots, consistent with the prediction of two-state association and dissociation. (ii) k(on) is independent of the nature of the base N located in the center of the third strand. (iii) k(on) strongly decreases when the NaCl concentration is decreased. (iv) The activation energy, E(on), is always negative and becomes more negative when the NaCl concentration is decreased. (v) k(off) is independent of NaCl concentration but depends on the base N, with its magnitude following the order C > G > A >> T. (vi) The activation energy, E(off), is independent of the base N. All these results are discussed in the light of a nucleation-zipping model similar to that developed for the duplex-coil transitions [Craig, M. E., Crothers, D. M., & Doty, P. (1971) J. Mol. Biol. 62,383-401; Porschke, D., Eigen, M. (1971) J. Mol. Biol. 62, 361-381].