Hurlburt B K, Yanofsky C
Department of Biological Sciences, Stanford University, CA 95403-5020.
Nucleic Acids Res. 1992 Jan 25;20(2):337-41. doi: 10.1093/nar/20.2.337.
The 3-dimensional structure of the trp repressor, aporepressor, and repressor/operator complex have been described. The NH2-terminal arms of the protein, comprising approximately 12-14 residues, were not well resolved in any of these structures. Previous studies by Carey showed that the arms are required for full in vitro repressor activity. To examine the roles of the arms more fully we have removed codons 2-5 and 2-8 of the trpR gene and analyzed the resulting truncated repressors in vivo and in vitro. The delta 2-5 trp repressor was found to be approximately 25% as active as the wild type repressor in vivo. In in vitro equilibrium binding experiments, the delta 2-5 trp repressor was shown to be five-fold less active in operator binding. The rate of dissociation of the complex formed between the delta 2-5 trp repressor and operator was essentially the same as the rate of dissociation of the wild type trp repressor/operator complex. However association of the delta 2-5 trp repressor with operator was clearly defective. Since the NH2-terminal arms of the trp repressor appear to affect association predominantly they may play a role in facilitating non-specific association of repressor with DNA as repressor seeks its cognate operators. The delta 2-8 trp repressor was unstable in vivo and in vitro, suggesting that some portion of the NH2-terminal arm is required for proper folding of the remainder of the molecule.
色氨酸阻遏物、无辅基阻遏物以及阻遏物/操纵基因复合物的三维结构已被描述。该蛋白质的氨基末端臂由大约12 - 14个残基组成，在这些结构中均未得到很好的解析。凯里之前的研究表明，这些臂对于完整的体外阻遏物活性是必需的。为了更全面地研究这些臂的作用，我们去除了trpR基因的密码子2 - 5和2 - 8，并在体内和体外分析了产生的截短型阻遏物。发现δ2 - 5色氨酸阻遏物在体内的活性约为野生型阻遏物的25%。在体外平衡结合实验中，δ2 - 5色氨酸阻遏物在与操纵基因结合时的活性低五倍。δ2 - 5色氨酸阻遏物与操纵基因形成的复合物的解离速率与野生型色氨酸阻遏物/操纵基因复合物的解离速率基本相同。然而，δ2 - 5色氨酸阻遏物与操纵基因的结合明显存在缺陷。由于色氨酸阻遏物的氨基末端臂似乎主要影响结合，当阻遏物寻找其同源操纵基因时，它们可能在促进阻遏物与DNA的非特异性结合中发挥作用。δ2 - 8色氨酸阻遏物在体内和体外都不稳定，这表明氨基末端臂的某些部分对于分子其余部分的正确折叠是必需的。
