NONSPECIFIC INTERACTIONS OF ESCHERICHIA-COLI RNA POLYMERASE WITH NATIVE AND DENATURED DNA DIFFERENCES IN THE BINDING BEHAVIOR OF CORE AND HOLO ENZYME
NONSPECIFIC INTERACTIONS OF ESCHERICHIA-COLI RNA POLYMERASE WITH NATIVE AND DENATURED DNA DIFFERENCES IN THE BINDING BEHAVIOR OF CORE AND HOLO ENZYME
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DOI：
10.1021/bi00602a006
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发表时间：
1978-01-01
期刊：
影响因子：
2.9
通讯作者：
RECORD M T JR
中科院分区：
文献类型：
--
作者：
DEHASETH P L;LOHMAN T M;RECORD M T JR
E.用双链（ds）和单链（ss）DNA研究了大肠杆菌RNA聚合酶核心和全酶。这些相互作用的结合常数作为单价和/或二价阳离子浓度，温度或pH值等溶液变量的函数，从含有固定化DNA的小柱的蛋白质洗脱分析确定。这种技术，虽然尚未经验，被证明产生准确的结合常数的非特异性相互作用的乳糖阻遏物与双链DNA。观察到的结合常数（Kobsd）是非常敏感的功能的一价阳离子浓度的核心和全酶与双链DNA的相互作用。在不存在二价阳离子的情况下，衍生物-（d log Kobsd/d log [Na+]）为11 . 21 . ±-. 3为核心-ds DNA相互作用。因此，大约11和21低分子量离子被释放，在热力学意义上，在形成的holo-ds和core-ds复合物，分别。离子释放是这些非特异性相互作用的热力学驱动力，并导致复合物的稳定性随着单价离子浓度的降低而显著增加。可能的分子模型，占不同的盐敏感性的holo-ds和core-ds配合物进行了讨论。竞争性配体Mg ~（2+）对这些相互作用的影响也进行了研究。大量离子释放（约18单价离子）也伴随着全或核心聚合酶与ss DNA的相互作用。在研究的离子浓度范围内，holo-SS相互作用基本上强于核心-SS相互作用;聚合酶与SS DNA的相互作用一般强于酶与DS DNA的非特异性相互作用。RNA聚合酶与DNA的非特异性相互作用显然具有生理相关性。这不仅是合理的假设，蛋白质的相同区域参与特异性和非特异性的相互作用，但RNA聚合酶和DNA的非特异性相互作用可能发挥作用，在热力学和动力学意义上，在决定这种蛋白质的可用性，启动子结合和RNA链起始。因此，RNA聚合酶的非特异性相互作用对离子条件的强烈依赖性表明，离子浓度在转录控制中可能具有调节作用。
The nonspecific interactions of E. coli RNA polymerase core and holoenzyme were investigated with double-stranded (ds) and single-stranded (ss) DNA. Binding constants for these interactions as functions of such solution variables as monovalent and/or divalent cation concentration, temperature or pH were determined from analysis of the elution of the proteins from small columns containing immobilized DNA. This technique, although as yet empirical, was demonstrated to yield accurate binding constants for the nonspecific interaction of lac repressor with ds DNA. Observed binding constants (Kobsd) are extraordinarily sensitive functions of the monovalent cation concentration for the interactions of both core and holoenzyme with ds DNA. In the absence of divalent cations, the derivatives -(d log Kobsd/d log [Na+]) are 11 .+-. 2 for the holo-ds DNA interaction and 21 .+-. 3 for the core-ds DNA interaction. Consequently, about 11 and 21 low-MW ions are released, in the thermodynamic sense, in the formation of the holo-ds and core-ds complexes, respectively. Ion release is a thermodynamic driving force for these nonspecific interactions and causes the stability of the complexes to increase very substantially with a reduction in monovalent ion concentration. Possible molecular models which account for the different salt sensitivities of the holo-ds and core-ds complexes are discussed. Effects of the competitive ligand Mg2+ on these interactions are also examined. Substantial ion release (.apprx. 18 monovalent ions) also accompanies the interaction of either holo or core polymerase with ss DNA. Over the range of ion concentrations investigated, the holo-ss interaction is substantially stronger than the core-ss interaction; the interactions of polymerase with ss DNA are, in general, stronger than the nonspecific interactions of the enzyme with ds DNA. The nonspecific interactions of RNA polymerase with DNA apparently have physiological relevance. Not only is it plausible to assume that the same regions of the protein are involved in both specific and nonspecific interactions, but nonspecific interactions of RNA polymerase and DNA may play a role in determining the availability of this protein, in both the thermodynamic and the kinetic sense, for promoter binding and RNA chain initiation. Consequently, the strong dependences of the nonspecific interactions of RNA polymerase on ionic conditions suggest the possibility of an modulating role of ion concentrations in the control of transcription.