DNA interaction and phosphotransfer of the C4-dicarboxylate-responsive DcuS-DcuR two-component regulatory system from Escherichia coli
DNA interaction and phosphotransfer of the C4-dicarboxylate-responsive DcuS-DcuR two-component regulatory system from Escherichia coli
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DOI：
10.1128/jb.186.6.1879-1889.2004
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发表时间：
2004-03-01
影响因子：
3.2
通讯作者：
Andrews, SC
中科院分区：
文献类型：
--
作者：
Abo-Amer, AE;Munn, J;Andrews, SC
大肠杆菌的DcuS-DcuR系统是一个双组分传感器-调节器，其响应于外部C-4-二羧酸和柠檬酸来控制基因表达。DcuS蛋白是特别有趣的，因为它含有两个PAS结构域，即周质C-4-二羧酸敏感PAS结构域（PASp）和胞质PAS结构域（PASc）的不确定功能。为了研究PASc结构域的作用，过量产生并检查了DcuS的三个不同片段：PASc-激酶、PASc和激酶。两个激酶结构域的片段被[γ-P-32]ATP自磷酸化。该速率不受富马酸盐或琥珀酸盐的影响，支持PASp结构域在C-4-二羧酸传感中的作用。两种磷酸化的DcuS构建体都能够将其磷酰基快速传递给DcuR，并且在磷酸化后，DcuR快速去磷酸化。没有发现与任何一种含PASc蛋白相关的辅基或大量金属。用纯化的DcuR蛋白研究其DNA结合特异性。它被发现从一个单体转化为二聚体乙酰磷酸处理后，和天然聚丙烯酰胺凝胶电泳表明，它可以寡聚化。DcuR特异性结合三种已知的DcuSR调节基因（dctA、dcuB和frdA）的启动子，未处理的DcuR的表观K（D）为6至32 μ M，乙酰磷酸处理的形式的表观K（D）小于或等于1至2 μ M。通过DNA酶I足迹法定位结合位点，从而鉴定推定的DcuR结合基序[串联重复的（T/A）（A/T）（T/C）（A/T）AA序列]。DcuR结合位点的dcuB，dctA，和frdA基因分别位于27，94，和86 bp，上游相应的+1位点，并确定了一个新的启动子dcuB，响应DcuR。
The DcuS-DcuR system of Escherichia coli is a two-component sensor-regulator that controls gene expression in response to external C-4-dicarboxylates and citrate. The DcuS protein is particularly interesting since it contains two PAS domains, namely a periplasmic C-4-dicarboxylate-sensing PAS domain (PASp) and a cytosolic PAS domain (PASc) of uncertain function. For a study of the role of the PASc domain, three different fragments of DcuS were overproduced and examined: they were PASc-kinase, PASc, and kinase. The two kinase-domain-containing fragments were autophosphorylated by [gamma-P-32]ATP. The rate was not affected by fumarate or succinate, supporting the role of the PASp domain in C-4-dicarboxylate sensing. Both of the phosphorylated DcuS constructs were able to rapidly pass their phosphoryl groups to DcuR, and after phosphorylation, DcuR dephosphorylated rapidly. No prosthetic group or significant quantity of metal was found associated with either of the PASc-containing proteins. The DNA-binding specificity of DcuR was studied by use of the pure protein. It was found to be converted from a monomer to a dimer upon acetylphosphate treatment, and native polyacrylamide gel electrophoresis suggested that it can oligomerize. DcuR specifically bound to the promoters of the three known DcuSR-regulated genes (dctA, dcuB, and frdA), with apparent K(D)s of 6 to 32 muM for untreated DcuR and less than or equal to1 to 2 muM for the acetylphosphate-treated form. The binding sites were located by DNase I footprinting, allowing a putative DcuR-binding motif [tandemly repeated (T/A)(A/T)(T/C)(A/T)AA sequences] to be identified. The DcuR-binding sites of the dcuB, dctA, and frdA genes were located 27, 94, and 86 bp, respectively, upstream of the corresponding +1 sites, and a new promoter was identified for dcuB that responds to DcuR.