Hurlburt B K, Yanofsky C
Department of Biological Sciences, Stanford University, California 94305.
J Biol Chem. 1993 Jul 15;268(20):14794-8.
The trp repressor of Escherichia coli is a dimeric DNA-binding protein that regulates transcription of several operons concerned with tryptophan metabolism. Although heterodimer formation between mutant and wild type subunits occurs readily in vivo, comparable heterodimers could be formed in vitro only under extreme conditions. To explain this difference we analyzed trp repressor dimer formation and dissociation using an in vitro transcription/translation system. Nascent wild type or mutant repressor polypeptides, synthesized in the presence of an excess of a second repressor, were invariably incorporated into heterodimers. In contrast, previously synthesized and assembled wild type dimers appeared to be refractory to dissociation, since they did not form heterodimers. However, previously synthesized mutant dimeric repressors that were defective in tryptophan binding readily dissociated and formed heterodimers. We noted that the ability of a dimeric repressor to dissociate under our conditions correlated inversely with its affinity for tryptophan. Consistent with this conclusion, we found that dissociation of the wild type aporepressor (no added tryptophan) was appreciably more rapid than dissociation of the tryptophan-saturated wild type repressor.
大肠杆菌的色氨酸阻遏物是一种二聚体DNA结合蛋白，它调控着几个与色氨酸代谢相关的操纵子的转录。虽然突变体亚基和野生型亚基之间的异源二聚体在体内很容易形成，但只有在极端条件下，才能在体外形成类似的异源二聚体。为了解释这种差异，我们使用体外转录/翻译系统分析了色氨酸阻遏物二聚体的形成和解离。在存在过量的另一种阻遏物的情况下合成的新生野生型或突变型阻遏物多肽总是会被整合到异源二聚体中。相比之下，先前合成并组装好的野生型二聚体似乎难以解离，因为它们不会形成异源二聚体。然而，先前合成的在色氨酸结合方面有缺陷的突变型二聚体阻遏物很容易解离并形成异源二聚体。我们注意到，在我们的条件下，二聚体阻遏物的解离能力与其对色氨酸的亲和力呈负相关。与这一结论一致，我们发现野生型无辅阻遏物（未添加色氨酸）的解离明显比色氨酸饱和的野生型阻遏物的解离要快。
