Kim J H, Chambliss G H
Department of Bacteriology, University of Wisconsin-Madison, E. B. Fred Hall, Madison, WI 53706, USA.
Nucleic Acids Res. 1997 Sep 1;25(17):3490-6. doi: 10.1093/nar/25.17.3490.
Catabolite control protein A (CcpA) is a global regulatory protein involved in catabolite repression and glucose activation in Gram-positive bacteria. cis -Acting DNA sequences, catabolite response elements ( cre s), involved in this regulatory system contain a 14 base pair (bp) region of dyad symmetry. CcpA, a repressor of the Lac I family, has been shown to bind specifically to cre s. To better understand cre recognition by CcpA, we have focused on the interaction between CcpA and the amyE cre , called amyO, which is located at the transcription start site of the alpha-amylase gene. DNA-protein complexes were probed with dimethylsulfate (DMS) and N -ethylnitrosourea (EtNU) to identify guanines and phosphates that participate in complex formation. Interaction between amyO and CcpA visualized through methylation protection and interference showed that CcpA contacts guanine residues at the outer bounds of amyO with higher affinity than near the dyad axis. From ethylation interference studies, it was found that CcpA contacts three phosphate groups at each end of amyO, and one or two phosphate groups near the dyad axis. Exonuclease III protection revealed that CcpA protects a 26 bp region centered around the dyad axis of amyO. The isolated N-terminal fragment still specifically bound to the sequence resembling the half sites of the amyO sequence. Considering these findings and the helical structure of B-DNA, our results suggest that each of the two monomers of the CcpA molecule contact the major groove in each half of the region of dyad symmetry and that the contacts are on the same face of the DNA helix, which is typical of bacterial repressor-operator interactions. However, the absence of strong contacts near the dyad axis by CcpA is in contrast to the situation with the gal repressor, another member of the Lac I family of repressors.
分解代谢物控制蛋白A（CcpA）是一种全局调节蛋白，参与革兰氏阳性菌中的分解代谢物阻遏和葡萄糖激活。参与该调节系统的顺式作用DNA序列，即分解代谢物反应元件（cre s），包含一个14个碱基对（bp）的二元对称区域。CcpA是Lac I家族的一种阻遏物，已被证明能特异性结合cre s。为了更好地理解CcpA对cre的识别，我们重点研究了CcpA与位于α-淀粉酶基因转录起始位点的amyE cre（称为amyO）之间的相互作用。用硫酸二甲酯（DMS）和N - 乙基亚硝基脲（EtNU）探测DNA - 蛋白质复合物，以鉴定参与复合物形成的鸟嘌呤和磷酸基团。通过甲基化保护和干扰观察到的amyO与CcpA之间的相互作用表明，CcpA与amyO外侧边界的鸟嘌呤残基接触的亲和力高于二元轴附近。从乙基化干扰研究中发现，CcpA与amyO两端的三个磷酸基团以及二元轴附近的一个或两个磷酸基团接触。核酸外切酶III保护显示，CcpA保护围绕amyO二元轴的一个26 bp区域。分离出的N端片段仍能特异性结合类似于amyO序列半位点的序列。考虑到这些发现以及B - DNA的螺旋结构，我们的结果表明，CcpA分子的两个单体各自与二元对称区域每一半的大沟接触，且接触位于DNA螺旋的同一面上，这是细菌阻遏物 - 操纵子相互作用的典型特征。然而，CcpA在二元轴附近缺乏强烈接触，这与Lac I家族阻遏物的另一个成员——半乳糖阻遏物的情况不同。
