Rolfes R J, Zalkin H
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
J Biol Chem. 1988 Dec 25;263(36):19653-61.
The Escherichia coli gene purR, encoding a repressor protein, was cloned by complementation of a purR mutation. Gene purR on a multicopy plasmid repressed expression of purF and purF-lacZ and reduced the growth rate of host cells by limiting the rate of de novo purine nucleotide synthesis. The level of a 1.3-kilobase purR mRNA was higher in cells grown with excess adenine, suggesting that synthesis of the repressor may be regulated. The chromosomal locus of purR was mapped to coordinate 1755-kb on the E. coli restriction map (Kohara, Y., Akiyama, K., and Isono, K. (1987) Cell 50, 495-508). Pur repressor bound specifically to purF operator DNA as determined by gel retardation and DNase I footprinting assays. The amino acid sequence of Pur repressor was derived from the nucleotide sequence. Pur repressor subunit contains 341 amino acids and has a calculated Mr of 38,179. Pur repressor is 31-35% identical with the galR and cytR repressors and 26% identical with the lacI repressor. These four repressors are likely homologous. Amino acid sequence similarity is greatest in an amino-terminal region presumed to contain a DNA-binding domain. A similarity is also noted in the operator sites for these repressors.
通过对嘌呤调节基因(purR)突变进行互补,克隆了编码阻遏蛋白的大肠杆菌基因purR。多拷贝质粒上的purR基因抑制了嘌呤磷酸核糖焦磷酸合成酶基因(purF)和purF - 乳糖操纵子融合基因(purF - lacZ)的表达,并通过限制嘌呤核苷酸从头合成的速率降低了宿主细胞的生长速度。在过量腺嘌呤存在下生长的细胞中,1.3千碱基的purR信使核糖核酸(mRNA)水平较高,这表明阻遏蛋白的合成可能受到调控。purR的染色体定位在大肠杆菌限制酶切图谱上被定位到1755千碱基的坐标处(小原洋、秋山和夫、矶野和男(1987年)《细胞》50卷,495 - 508页)。通过凝胶阻滞分析和脱氧核糖核酸酶I足迹分析确定,嘌呤阻遏蛋白特异性结合到purF操纵基因DNA上。嘌呤阻遏蛋白的氨基酸序列由核苷酸序列推导得出。嘌呤阻遏蛋白亚基包含341个氨基酸,计算得出的分子量为38179。嘌呤阻遏蛋白与半乳糖操纵子阻遏蛋白(galR)和胞嘧啶代谢阻遏蛋白(cytR)有31% - 35%的同源性,与乳糖操纵子阻遏蛋白(lacI)有26%的同源性。这四种阻遏蛋白可能是同源的。在推测包含DNA结合结构域的氨基末端区域,氨基酸序列相似性最大。在这些阻遏蛋白的操纵基因位点也发现了相似性。