Characterization of the activating region of Escherichia coli catabolite gene activator protein (CAP). I. Saturation and alanine-scanning mutagenesis.
Characterization of the activating region of Escherichia coli catabolite gene activator protein (CAP). I. Saturation and alanine-scanning mutagenesis.
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DOI：
10.1016/0022-2836(94)90034-5
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发表时间：
1994-11
影响因子：
5.6
通讯作者：
Wei Niu;Yuhong Zhou;Qianping Dong;Y. Ebright;R. Ebright
中科院分区：
文献类型：
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作者：
Wei Niu;Yuhong Zhou;Qianping Dong;Y. Ebright;R. Ebright
大肠杆菌（Escherichia coli）分解代谢物基因激活蛋白（catabolite gene activator protein，CAP）的第152 - 166位氨基酸残基组成的表面环与启动子上的RNA聚合酶直接蛋白质-蛋白质接触。在这项工作中，我们已经使用了有针对性的饱和诱变的密码子152至166的编码CAP的基因，随后通过筛选，分离出200多个独立的突变体的CAP缺陷的转录激活，但没有缺陷的DNA结合。所有分离的单取代突变体仅映射到8个氨基酸残基：156、157、158、159、160、162、163和164。我们建议，这些残基定义的全部范围内的表位CAP的建议CAP-RNA聚合酶的相互作用。此外，我们在CAP的152 - 166位残基的每个位置构建了丙氨酸取代，并分析了对启动子转录激活和DNA结合的影响。丙氨酸取代Thr 158导致转录激活中约8倍的特异性缺陷。相比之下，丙氨酸取代没有其他残基测试结果在转录激活的两倍以上的特定缺陷。我们的结论是Thr 158的Cβ以外的侧链原子是lacpromoter转录激活所必需的，我们认为Thr 158的OHγ在lacpromoter的三元复合物中与RNA聚合酶直接接触。CAP和RNA聚合酶。我们进一步得出结论，除了Thr 158以外，没有其他残基是Cβ以外的侧链原子对lac启动子的转录激活是必需的。
It has been proposed that the surface loop consisting of amino acid residues 152 to 166 of the catabolite gene activator protein (CAP) ofEscherichia colimakes direct protein-protein contact with RNA polymerase at thelacpromoter. In this work, we have used targeted saturation mutagenesis of codons 152 to 166 of the gene encoding CAP, followed by a screen, to isolate more than 200 independent mutants of CAP defective in transcription activation but not defective in DNA binding. All isolated single-substitution mutants map to just eight amino acid residues: 156, 157, 158, 159, 160, 162, 163 and 164. We propose that these residues define the full extent of the epitope on CAP for the proposed CAP-RNA polymerase interaction. In addition, we have constructed alanine substitutions at each position from residue 152 to 166 of CAP, and we have analyzed the effects on transcription activation at thelacpromoter and on DNA binding. Alanine substitution of Thr 158 results in an approximately eightfold specific defect in transcription activation. In contrast, alanine substitution of no other residue tested results in a more than twofold specific defect in transcription activation. We conclude that, for Thr158, side-chain atom beyond Cβare essential for transcription activation at thelacpromoter, and we propose that Thr158 OHγmakes direct contact with RNA polymerase in the ternary complex forlacpromoter. CAP and RNA polymerase. We conclude further that for no residue other than Thr158 are side-chain atoms beyond Cβessential for transcription activation at thelacpromoter.