MOLECULAR-PARAMETERS CHARACTERIZING INTERACTION OF ESCHERICHIA-COLI LAC REPRESSOR WITH NON-OPERATOR DNA AND INDUCER
MOLECULAR-PARAMETERS CHARACTERIZING INTERACTION OF ESCHERICHIA-COLI LAC REPRESSOR WITH NON-OPERATOR DNA AND INDUCER
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DOI：
10.1021/bi00641a001
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发表时间：
1977-01-01
期刊：
影响因子：
2.9
通讯作者：
VONHIPPEL, PH
中科院分区：
文献类型：
--
作者：
BUTLER, AP;REVZIN, A;VONHIPPEL, PH
描述了非操纵子DNA和诱导剂与Lac抑制物结合的化学计量学，以及这些相互作用的构象方面。抑制子-非操纵子DNA复合体的圆二色谱(CD)与通过将单独组分的光谱相加而获得的谱有明显不同；主要变化是阳性(.apprx)的显著增强。275 nm)，原本保守的DNA B形成图案。这些CD光谱的变化似乎反映了抑制物结合上DNA构象的变化，并被解释为天然结构的一些碱基对的倾斜(相对于DNA轴)，或者可能是整体结构的一些扭曲导致邻近碱基转移矩的更紧密耦合。在紧密结合的条件下，监测非操纵子DNA或抑制物结合的CD谱中的这些变化作为添加的阻遏物浓度的函数，以确定与非操纵子DNA结合的位置大小(N)为.apprx。12个碱基对/抑制物四聚体(apprx.如果抑制子与双螺旋DNA晶格的两侧结合，则为24个碱基对)。根据结合等温线的计算和抑制分子SH基团的滴定作为添加DNA浓度的函数，证实了n的这一值。根据抑制子的操纵子和非操纵子DNA结合之间的关系，以及已知操纵子序列的各种特征，讨论了所测得的位点大小，以提出阻遏子-操纵子相互作用的几何模型。用平衡透析、荧光和凝胶渗透层析法研究阻遏剂-诱导剂的相互作用，结果表明，不同的阻遏剂制剂表现出不同的活性诱导剂结合位点数，每个阻遏剂四聚体(Ni)的平均数目不同；nI值在2到4之间。考察了不同环境条件对Ni的影响，并根据阻遏亚基与诱导物强结合而与操纵子弱结合的形式(RI)和与操纵子强结合而与诱导物弱结合的形式(RO)之间的构象平衡，与文献中的相关数据进行了讨论。还测定了诱导剂与阻遏亚基(RI形式)结合的热力学参数。在pH 7.6(4-25℃)。C)、差额学位。[标准自由能]=-7.7千卡/摩尔(25度C)、差额学位。[标准热焓]=-6.2千卡/摩尔，差S度。[标准熵]=+5卡·摩尔~(-1)度~(-1)。
The stoichiometries of binding of non-operator DNA and inducer to lac repressor, and some conformational aspects of these interactions, are described. The circular dichroism (CD) spectrum of the repressor-non-operator DNA complex is appreciably different from that obtained by summing the spectra of the separate components; the major change is a substantial enhancement of the positive (.apprx. 275 nm) lobe of the originally conservative DNA B form pattern. These CD spectral changes appear to reflect a change in DNA conformation on repressor binding and are interpreted in terms of tilting (relative to the DNA axis) of some of the base pairs of the native structure, or perhaps some twisting of the overall structure resulting in a tighter coupling of vicinal base transition moments. These changes in the CD spectrum of non-operator DNA or repressor binding were monitored as a function of added repressor concentration, under tight-binding conditions, to establish that the site size (n) for binding to non-operator DNA is .apprx. 12 base pairs/repressor tetramer (.apprx. 24 base pairs if repressor binds to both sides of the double helical DNA lattice). This value of n is confirmed both by calculation from binding isotherms and by titration of repressor SH groups as a function of added DNA concentration. The measured site size is discussed in terms of the relationship between operator and non-operator DNA binding of repressor, and of various features of the known operator sequence, to suggest alternative models for the geometry of the repressor-operator interaction. Investigation of the repressor-inducer interaction by equilibrium dialysis, fluorescence and gel permeation chromatography shows, in confirmation of other results, that different repressor preparations exhibit different (average) numbers of active inducer binding sites per repressor tetramer (nI); values of nI between 2 and 4 were obtained. The effects on nI of a variety of environmental conditions were examined, and the results, together with relevant data from the literature, are discussed in terms of conformational equilibria between forms of repressor subunits which bind strongly to inducer and weakly to operator (RI), and forms which bind strongly to operator and weakly to inducer (RO). Thermodynamic parameters for the binding of inducer to repressor subunits (in the RI form) were also determined. At pH 7.6 (4-25.degree. C), .DELTA.G.degree. [standard free energy] = -7.7 kcal/mol (25.degree. C), .DELTA.H.degree. [standard enthalpy] = -6.2 kcal/mol, and .DELTA.S.degree. [standard entropy] = +5 cal mol-1 deg-1 for this reaction.