REGULATION OF THE ESCHERICHIA-COLI L-ARABINOSE OPERON STUDIED BY GEL-ELECTROPHORESIS DNA-BINDING ASSAY
REGULATION OF THE ESCHERICHIA-COLI L-ARABINOSE OPERON STUDIED BY GEL-ELECTROPHORESIS DNA-BINDING ASSAY
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DOI：
10.1016/0022-2836(84)90241-9
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发表时间：
1984-01-01
影响因子：
5.6
通讯作者：
SCHLEIF, RF
中科院分区：
文献类型：
--
作者：
HENDRICKSON, W;SCHLEIF, RF
用聚丙烯酰胺凝胶电泳法测定了大肠杆菌l -阿拉伯糖操纵子诱导所需蛋白质的DNA结合特性。通过观察RNA聚合酶、cAMP受体蛋白和araC蛋白与含有araC或araBAD启动子区的短DNA片段的多重相互作用，研究了诱导和抑制机制。这些研究表明，araC蛋白与操作位点araO1的结合直接阻断了araC启动子pc上RNA聚合酶的结合。cAMP受体蛋白和araC蛋白不能在各自的位点上与含有pBAD启动子的线性DNA片段合作结合。然而，这两种积极效应必须存在于DNA上，以刺激RNA聚合酶的结合。蛋白质与DNA的结合是不够的；araC蛋白也必须处于诱导状态，RNA聚合酶才能结合。确定了araC蛋白与araI和araO1位点的平衡结合约束和动力学。在诱导剂l -阿拉伯糖的存在下，araC蛋白与含有任一位点的DNA片段结合具有相同的亲和力。在抗诱导剂D-聚焦的存在下，对这两个位点的亲和力降低了40倍。蛋白质两种状态的表观平衡结合常数与缓冲盐浓度平行变化。这一结果表明，araC蛋白的诱导和抑制形式在结合DNA时取代了相似数量的阳离子。
DNA binding properties of the proteins required for induction of the E. coli L-arabinose operon were measured using a polyacrylamide gel electrophoresis assay. The mechanisms of induction and repression were studied by observing the multiple interactions of RNA polymerase, cAMP receptor protein and araC protein with short DNA fragments containing either the araC or araBAD promoter regions. These studies show that binding of araC protein to the operator site,araO1, directly blocks RNA polymerase binding at the araC promoter, pc. The cAMP receptor protein and araC protein do not bind co-operatively at their respective sites to linear DNA fragments containing the pBAD promoter. Nevertheless, both these positive effectors must be present on the DNA to stimulate binding of RNA polymerase. Binding of the proteins to the DNA is not sufficient; araC protein must also be in the inducing state for RNA polymerase to bind. Equilibrium binding constraints and kinetics were determined for araC protein binding to the araI and the araO1 sites. In the presence of inducer, L-arabinose, araC protein binds with equal affinity to DNA fragments containing either site. In the presence of anti-inducer, D-fucose, the affinity for both sites is reduced 40-fold. The apparent equilibrium binding constants for both states of the protein vary in parallel with the buffer salt concentration. This result suggests that the inducing and repressing forms of araC protein displace a similar number of cations upon binding DNA.