Role of protein--protein interactions in the regulation of transcription by trp repressor investigated by fluorescence spectroscopy.
Role of protein--protein interactions in the regulation of transcription by trp repressor investigated by fluorescence spectroscopy.
复制标题
通过荧光光谱研究蛋白质-蛋白质相互作用在trp阻遏物转录调节中的作用。
DOI：
10.1021/bi00128a018
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Royer,C
中科院分区：
文献类型：
--
作者：
Fernando,T;Royer,C
威斯康星大学麦迪逊分校药学院，地址：425 North Charter，麦迪逊，威斯康星州53706;伊利诺伊大学生物化学系，地址：1209 West加州，Urbana，Illinois 61801，接收日期：1991年10月14日;修订后的Mandatapt，接收日期：1992年1月31日摘要：在目前的工作中，我们的特点是蛋白质-蛋白质相互作用的色氨酸阻遏物（TR）从大肠杆菌使用荧光光谱。用5-（二甲氨基）萘-1-磺酰胺（DNS）标记阻遏物，利用其稳态和时间分辨荧光各向异性来监测辅阻遏物存在和不存在时的亚基平衡。在不存在色氨酸的情况下，在所研究的浓度范围内（二聚体中约0.04-40 μ M），抑制剂在四聚体和二聚体之间处于平衡。辅阻遏物的结合导致四聚体的显著不稳定。二聚体-单体解离转变的开始通过监测在二聚体中稀释低于0.1 mM时固有色氨酸发射能量的降低来观察，表明二聚体解离常数的上限接近1 nM。用含有trp EDCBA操纵基因的26个碱基对的DNA滴定，在有和无辅阻遏物的情况下，与色氨酸存在时的1：1二聚体/操纵基因化学计量一致，而辅阻遏物与TR二聚体/DNA化学计量大于1结合，这取决于蛋白质和操纵基因的浓度。利用荧光中的多个可观察参数，我们对这种细菌阻遏物中的偶联平衡进行了彻底的研究。我们的研究结果是一致的生理相关的热力学作用，四聚体的色氨酸阻遏物的调节功能。目前的研究结果揭示了色氨酸阻遏物系统中新的蛋白质-蛋白质相互作用，这表明荧光光谱方法在研究真核系统中蛋白质-蛋白质相互作用的作用方面也是非常有用的。蛋白质-蛋白质相互作用已经成为控制转录调节的基本一般机制之一。这种蛋白质寡聚化相互作用参与调节原核阻遏物与DNA的结合，所述原核阻遏物例如arc（Bowie & Sauer，1989）、lambda（Senear & Ackers，1990）和lac阻遏物（Royer等人，1990年）。差异二聚化亲和力参与癌基因产物激活真核转录。这项工作得到了美国国立卫生研究院（R-29-GM 39969）对CAR的资助。通信地址 *伊利诺伊大学 *威斯康星大学麦迪逊分校fos和jun（Turner和Tijan，1989; Gentz等，一九八九年; Kouzerides和Ziff，1989），其也通过蛋白质-蛋白质相互作用在调节激素受体的活性中发挥作用（Diamond等人，1990年）。已经鉴定了涉及发育和分化的许多螺旋-环-螺旋型蛋白质的正和负调节二聚化配偶体（Baringa，1991;黑木& Eisenman，1991; Prendergast et al.，1991年）。已经解决了几种DNA结合蛋白的三维结构，无论是单独的还是与它们的同源DNA序列复合的，包括trp阻遏物的那些（Schevitz et al.，1985; Zhang等人，1987; Otwinowski等人，1988年），这些提供了一个框架，了解他们的功能。然而，一个完整的理解物理-
School of Pharmacy, University of Wisconsin—Madison, 425 North Charter, Madison, Wisconsin 53706, and Department of Biochemistry, University of Illinois, 1209 West California, Urbana, Illinois 61801 Received October 14, 1991; Revised Manuscript Received January 31, 1992 abstract: In the present work, we have characterized the protein-protein interactions in the trp repressor (TR) from Escherichia coli using fluorescence spectroscopy. The steady-state and time-resolved fluorescence anisotropy of repressor labeled with 5-(dimethylamino) naphthalene-l-sulfonamide (DNS) was used to monitor subunit equilibria in the absence and presence of corepressor. In the absence of tryptophan, therepressor is in equilibrium between tetramers and dimers in the concentration range studied (approximately 0.04-40 juM in dimer). Binding of corepressor resulted in a marked destabilization of the tetramer. The beginning of a dimer-monomer dissociation transition was observed by monitoring the decrease in the intrinsic tryptophan emission energy upon dilution below 0.1 mM in dimer, indicating an upper limit for the dimer-dissociation constant near 1 nM. DNA titrations with a 26 base pair sequence containing the trp EDCBA operator performed in the absence and presence of the corepressor are consistent with a 1: 1 dimer/operator stoi-chiometry in the presence of tryptophan, while theaporepressor binds with TR dimer/DNA stoichiometries greater than one and which depend upon both the concentration of protein and that of the operator. Using the multiple observable parameters available in fluorescence, we have thuscarried out a thorough investigation of the coupled equilibria in this bacterial repressor. Our results are consistent with a physiologically relevant thermodynamic role for tetramerization in the regulatory function of the trp repressor. The present results which have brought to light novel protein-protein interactions in the trp repressor system indicate that fluorescence spectroscopic methods could prove quite useful in the study of the role of protein-protein interactions in eukaryotic systems as well.^^ otein-protein interactions have emerged as one of the underlying general mechanisms governing the regulation of transcription. Such protein oligomerization interactions in-tervene in regulating the binding to DNA by prokaryotic repressors such as the arc (Bowie & Sauer, 1989), lambda (Senear & Ackers, 1990), and lac repressors (Royer et al., 1990). Differential dimerization affinities are involved in the activation of eukaryotic transcription by the oncogene products tThis work was supported by a grant to CAR from the National Institutes of Health (R-29-GM39969).* To whom correspondence shouldbe addressed.* University of Illinois.* University of Wisconsin—Madison. fos and jun (Turner & Tijan, 1989; Gentz et al., 1989; Kouzerides & Ziff, 1989), which also play a role through protein-protein interactions in modulating the activity of hormonal receptors (Diamond et al., 1990). Both positive and negative regulating dimerization partners have been identified for a number of helix-loop-helix-type proteins implicated in development and differentiation (Baringa, 1991; Blackwood & Eisenman, 1991; Prendergast et al., 1991). The three-dimensional structure for several DNA-binding proteins, both alone and complexed with their cognate DNA sequences, have been solved, including those of the trp repressor (Schevitz et al., 1985; Zhang et al., 1987; Otwinowski et al., 1988), and these provide the framework for an understanding of their function. However, a complete understanding of the physi-