Quantitative analysis of the ternary complex of RNA polymerase, cyclic AMP receptor protein and DNA by fluorescence anisotropy measurements
Quantitative analysis of the ternary complex of RNA polymerase, cyclic AMP receptor protein and DNA by fluorescence anisotropy measurements
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DOI：
10.18388/abp.2008_3060
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发表时间：
2008-01-01
影响因子：
1.7
通讯作者：
Wasylewski, Zygmunt
中科院分区：
文献类型：
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作者：
Bonarek, Piotr;Kedracka-Krok, Sylwia;Wasylewski, Zygmunt
在体外形成的转录复合物与大肠杆菌RNA聚合酶进行了监测，使用荧光各向异性测量标记的DNA片段。多组分系统由全或核心RNA聚合酶（RNAP）和DNA的lac或gal启动子片段（以不同的构型）组成，在存在或不存在CRP激活蛋白（wt或突变体）及其配体cAMP的情况下。表征该系统的表观结合常数的值，作为在该系统中发生的所有过程的结果。在不存在CRP蛋白的情况下，启动子与核心RNAP的相互作用的特征在于lac 166和gal 178的表观结合常数分别为0.67和1.9 × 10（6）M-1，并且可以被认为是非特异性的。wt CRP的存在增强了核心RNAP与启动子的相互作用的强度，并且甚至在gal启动子的情况下，它使这种相互作用具有特异性（表观结合常数2.93 X 10（7）M-1）。全RNAP与启动子的结合明显强于核心RNAP（lac 166和gal 178的表观结合常数分别为1.46和40.14 × 10（6）M-1），CRP的存在也增强了这些相互作用的强度。CRP激活区1的突变没有引起任何显着的干扰，在全息RNAP乳糖启动子的相互作用，但激活蛋白的激活区2的突变大大削弱了RNAP半乳糖启动子的相互作用。
The in vitro formation of transcription complexes with Escherichia coli RNA polymerase was monitored using fluorescence anisotropy measurements of labeled fragments of DNA. The multicomponent system consisted of holo or core RNA polymerase (RNAP) and lac or gal promoter fragments of DNA (in different configurations), in the presence or absence of CRP activator protein (wt or mutants) with its ligand, cAMP. Values of the apparent binding constants characterizing the system were obtained, as a result of all processes taking place in the system. The interaction of the promoters with core RNAP in the absence of CRP protein was characterized by apparent binding constants of 0.67 and 1.9X10(6) M-1 for lac166 and gal178, respectively, and could be regarded as nonspecific. The presence of wt CRP enhanced the strength of the interaction of core RNAP with the promoter, and even in the case of gal promoter it made this interaction specific (apparent binding constant 2.93 X 10(7) M-1). Holo RNAP bound the promoters significantly more strongly than core RNAP did (apparent binding constants 1.46 and 40.14 x 10(6) M-1 for lac166 and gal178, respectively), and the presence of CRP also enhanced the strength of these interactions. The mutation in activator region 1 of CRP did not cause any significant disturbances in the holo RNAP-lac promoter interaction, but mutation in activator region 2 of the activator protein substantially weakened the RNAP-gal promoter interaction.