Snapyan Marina, Lecocq Michèle, Guével Laetitia, Arnaud Marie-Claire, Ghochikyan Anahit, Sakanyan Vehary
Laboratoire de Biotechnologie, FRE-CNRS 2230 Biocatalyse, Université de Nantes, Nantes, France.
Proteomics. 2003 May;3(5):647-57. doi: 10.1002/pmic.200300390.
The protein array methodology is used to study DNA-protein and protein-protein interactions governing gene expression from the Bacillus stearothermophilus PargCo promoter-operator region. Using probes labelled with near-infrared fluorescence dyes with exitation characteristics close to 700 or 800 nm, it is possible to detect signals from proteins (purified or non-purified in Escherichia coli cell extracts) immobilised on a nitrocellulose membrane with a high sensitivity (almost 12 amol of a spotted protein for protein-DNA interactions). Protein array data are confirmed by other methods indicating that molecular interactions of the order 10(-7) M can be monitored with the proposed protein array approach. We show that the PargCo region is a target for binding at least three types of regulatory proteins, ArgR repressors from thermophilic bacteria, the E. coli RNA polymerase alpha subunit and cyclic AMP binding protein CRP. We also demonstrate that the high strength of the PargC promoter is related to an upstream element that binds to the E. coli RNA polymerase alpha subunit.
蛋白质阵列方法用于研究嗜热脂肪芽孢杆菌PargCo启动子-操纵子区域中控制基因表达的DNA-蛋白质和蛋白质-蛋白质相互作用。使用标记有激发特性接近700或800 nm的近红外荧光染料的探针，可以高灵敏度地检测固定在硝酸纤维素膜上的蛋白质（在大肠杆菌细胞提取物中纯化或未纯化）发出的信号（对于蛋白质-DNA相互作用，斑点蛋白几乎为12 amol）。蛋白质阵列数据通过其他方法得到证实，表明所提出的蛋白质阵列方法可以监测10(-7) M量级的分子相互作用。我们表明，PargCo区域是至少三种类型调节蛋白的结合靶点，即嗜热细菌的ArgR阻遏物、大肠杆菌RNA聚合酶α亚基和环磷酸腺苷结合蛋白CRP。我们还证明，PargC启动子的高强度与一个与大肠杆菌RNA聚合酶α亚基结合的上游元件有关。
