大肠杆菌色氨酸操纵子阻遏蛋白的检测与分离
Detection and isolation of the repressor protein for the tryptophan operon of Escherichia coli.
作者信息
Zubay G, Morse D E, Schrenk W J, Miller J H
出版信息
Proc Natl Acad Sci U S A. 1972 May;69(5):1100-3. doi: 10.1073/pnas.69.5.1100.
Abstract
DNA from a transducing bacteriophage carrying a fusion of the tryptophan and lactose operons of E. coli (lambdadtrp-lac) has been used to direct cell-free synthesis of beta-galactosidase (EC 3.2.1.23). Whereas normal lac operon (lambdadlac) DNA requires adenosine-3':5'-cyclic monophosphate (cAMP) for beta-galactosidase synthesis, trp-lac DNA is unaffected by cAMP. This difference in cAMP dependence verifies the presence of a cAMP-requiring promoter in the lac operon that has been removed from the trp-lac DNA. Synthesis with trp-lac DNA is controlled by the protein product of the tryptophan repressor gene (trpR). Synthesis in extracts of trpR(-) (repressor-negative) cells is progressively reduced by increased additions of extract from trpR(+) cells. No trpR(-) product repression is seen when beta-galactosidase synthesis is programmed by normal lac DNA. This highly sensitive and specific assay has facilitated quantitation and partial purification of the trp repressor.
摘要
携带大肠杆菌色氨酸和乳糖操纵子融合基因（λdtrp-lac）的转导噬菌体的DNA已被用于指导β-半乳糖苷酶（EC 3.2.1.23）的无细胞合成。正常的乳糖操纵子（λdlac）DNA合成β-半乳糖苷酶需要3':5'-环磷酸腺苷（cAMP），而trp-lac DNA不受cAMP影响。这种对cAMP依赖性的差异证实了乳糖操纵子中存在一个需要cAMP的启动子，该启动子已从trp-lac DNA中去除。用trp-lac DNA进行的合成受色氨酸阻遏基因（trpR）的蛋白质产物控制。随着trpR(+)细胞提取物添加量的增加，trpR(-)（阻遏物阴性）细胞提取物中的合成逐渐减少。当由正常的乳糖操纵子DNA指导β-半乳糖苷酶合成时，未观察到trpR(-)产物的阻遏作用。这种高度灵敏和特异的检测方法有助于对色氨酸阻遏物进行定量和部分纯化。
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