DNA sequence determinants for binding of the Escherichia coli catabolite gene activator protein.
DNA sequence determinants for binding of the Escherichia coli catabolite gene activator protein.
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DOI：
10.1016/s0021-9258(18)42099-6
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发表时间：
1992-07
期刊：
影响因子：
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通讯作者：
A. Gunasekera;Y. Ebright;R. Ebright
中科院分区：
文献类型：
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作者：
A. Gunasekera;Y. Ebright;R. Ebright
大肠杆菌分解代谢基因激活蛋白(CAP)的DNA结合位点共有22个碱基对，具有2倍对称性：5‘-AAATGTGATCTAGATCACATTT-3’。共识DNA半位点的每一半的第4到8位是最保守的。在这份报告中，我们分析了4到8位DNA碱基对替换的影响，4、6和8位尿嘧啶和5-甲基胞嘧啶替换胸腺嘧啶的影响，以及7到10位5‘-GATC-3’序列DAM甲基化的影响。所有在4到8位DNA碱基对替换的DNA位点都显示出比共识DNA位点更低的与CAP的亲和力，这与共识DNA位点是CAP的理想DNA位点的说法一致。第4位T：A的特异性似乎仅由胸腺嘧啶5-甲基决定。T：A在第6位的特异性和A：T在第8位的特异性似乎部分但不完全由胸腺嘧啶5-甲基决定。DAM甲基化对CAP.DNA复合体的形成影响不大。热力学定义的共识DNA位点跨越28个碱基对。CAP的最大亲和力和热力学定义的最大CAP所需的所有或几乎所有DNA决定簇。DNA离子对的形成包含在一个28碱基对的DNA片段中，该DNA片段的中心是22个碱基对的共识DNA位点。本报告中的定量数据提供了详细研究蛋白质-DNA复合体中氨基酸-碱基对和氨基酸-磷酸接触所需的基线热力学数据。
The consensus DNA site for binding of the Escherichia coli catabolite gene activator protein (CAP) is 22 base pairs in length and is 2-fold symmetric: 5'-AAATGTGATCTAGATCACATTT-3'. Positions 4 to 8 of each half of the consensus DNA half-site are the most strongly conserved. In this report, we analyze the effects of substitution of DNA base pairs at positions 4 to 8, the effects of substitution of thymine by uracil and by 5-methylcytosine at positions 4, 6, and 8, and the effect of dam methylation of the 5'-GATC-3' sequence at positions 7 to 10. All DNA sites having substitutions of DNA base pairs at positions 4 to 8 exhibit lower affinities for CAP than does the consensus DNA site, consistent with the proposal that the consensus DNA site is the ideal DNA site for CAP. Specificity for T:A at position 4 appears to be determined solely by the thymine 5-methyl group. Specificity for T:A at position 6 and specificity for A:T at position 8 appear to be determined in part, but not solely, by the thymine 5-methyl group. dam methylation has little effect on CAP.DNA complex formation. The thermodynamically defined consensus DNA site spans 28 base pairs. All, or nearly all, DNA determinants required for maximal affinity for CAP and for maximal thermodynamically defined CAP.DNA ion pair formation are contained within a 28-base pair DNA fragment that has the 22-base pair consensus DNA site at its center. The quantitative data in this report provide base-line thermodynamic data required for detailed investigations of amino acid-base pair and amino acid-phosphate contacts in this protein-DNA complex.