Mutant Trp repressors with new DNA-binding specificities.
Mutant Trp repressors with new DNA-binding specificities.
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具有新 DNA 结合特异性的突变 Trp 阻遏蛋白。
DOI：
10.1126/science.3140377
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发表时间：
1988
期刊：
影响因子：
--
通讯作者：
Youderian,P
中科院分区：
文献类型：
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作者：
Bass,S;Sorrells,V;Youderian,P
对大肠杆菌色氨酸去阻遏物基因的谷氨酰胺68（Gln 68）、赖氨酸72（Lys 72）、异亮氨酸79（Ile 79）、丙氨酸80（Ala 80）和苏氨酸81（Thr 81）密码子进行寡核苷酸定向诱变，以制备具有36个不同氨基酸变化的突变阻遏物。测试突变阻遏物与一组28个不同操纵子的每个成员的结合，这些操纵子与共识操纵子密切相关。在36个突变阻遏物中，11个结合28个操纵子的子集;其中5个具有新的结合特异性。这些新的特异性表明，羟基的Thr 81使一个特定的接触与四个关键的碱基对在atropoperator半网站，和甲基的Thr 81决定特异性在第二个，关键的碱基对。Trp阻遏物不使用其“识别α-螺旋”的前两个氨基酸Ile 79和Ala 80来进行序列特异性DNA接触，并且在体内以与噬菌体λ阻遏物、λ Cro蛋白和大肠杆菌噬菌体434阻遏物接触其各自结合位点的方式根本不同的方式与其操纵基因相互作用。
Oligonucleotide-directed mutagenesis of the codons for glutamine-68 (Gln68), lysine-72 (Lys72), isoleucine-79 (Ile79), alanine-80 (Ala80), and threonine-81 (Thr81) of theEscherichia colitrpR(tryptophan aporepressor) gene was used to make mutant repressors with each of 36 different amino acid changes. Mutant repressors were tested for binding to each member of a set of 28 different operators closely related to the consensustrpoperator. Of the 36 mutant repressors, 11 bind a subset of the 28 operators; 5 of these have new binding specificities. These new specificities indicate that the hydroxyl group of Thr81makes a specific contact with one of the four critical base pairs in atrpoperator half-site, and the methyl group of Thr81determines specificity at a second, critical base pair. The Trp repressor does not use the first two amino acids of its "recognition α-helix," Ile79and Ala80, to make sequence-specific DNA contacts, and interacts with its operator in vivo in a way fundamentally different from the way that phage lambda repressor, lambda Cro protein, and coliphage 434 repressor contact their respective binding sites.