Kholti A, Charlier D, Gigot D, Huysveld N, Roovers M, Glansdorff N
Laboratoire de Microbiologie, Université Libre de Bruxelles, 1-av. E. Gryson, Brussels, B-1070, Belgium.
J Mol Biol. 1998 Jul 24;280(4):571-82. doi: 10.1006/jmbi.1998.1910.
The carAB operon of the enterics Escherichia coli K-12 and Salmonella typhimurium LT2, encoding the sole carbamoylphosphate synthetase (CPSase) of these organisms, is transcribed from two promoters in tandem, carP1 upstream and carP2 downstream, repressed respectively by pyrimidines and arginine. We present evidence that the pyrH gene product (the hexameric UMP-kinase) directly participates in the pyrimidine-specific control of carP1 activity. Indeed, we have isolated in E. coli a particular type of pyrH mutation (pyrH41) that retains a quasi-normal UMP-kinase activity, but yet is impaired in the pyrimidine-specific repression of the P1 promoter of the carAB operon of E. coli and of S. typhimurium. Moreover, the pyrimidine-dependent inhibition of in vivo Dam methylase modification of adenine -106 upstream of the carP1 promoter is altered in this pyrH mutant. The recessive pyrH41 allele bears a single C-G to A-T transversion that converts alanine 94 into glutamic acid (A94E). Although overexpression of pyrH41 results in UMP-kinase levels far above that of a wild-type strain, pyrimidine-specific repression of the carAB operon is not restored under these conditions. Similarly, overexpression of the UMP-CMP-kinase gene of Dictyostelium discoideum in the pyrH41 mutant does not restore pyrimidine-mediated control of carP1 promoter activity, in spite of the elevated UMP-kinase activity measured in such transformants. These results indicate that besides its catalytic function in the de novo pyrimidine biosynthesis, E. coli UMP-kinase fulfils an additional, but previously unrecognized role in the regulation of the carAB operon. UMP-kinase might function as the real sensor of the internal pyrimidine nucleotide pool and act in concert with the integration host factor (IHF) and aminopeptidase A (PepA alias CarP and XerB) in the elaboration of the complex nucleoprotein structure required for pyrimidine-specific repression of carP1 promoter activity.
肠道菌大肠杆菌K - 12和鼠伤寒沙门氏菌LT2的carAB操纵子，编码这些生物体唯一的氨甲酰磷酸合成酶（CPSase），由两个串联的启动子转录，上游的carP1和下游的carP2，分别受嘧啶和精氨酸抑制。我们提供证据表明，pyrH基因产物（六聚体UMP激酶）直接参与carP1活性的嘧啶特异性调控。实际上，我们在大肠杆菌中分离出一种特殊类型的pyrH突变（pyrH41），它保留了准正常的UMP激酶活性，但在大肠杆菌和鼠伤寒沙门氏菌carAB操纵子P1启动子的嘧啶特异性抑制方面存在缺陷。此外，在这个pyrH突变体中，carP1启动子上游腺嘌呤 - 106的体内Dam甲基化修饰的嘧啶依赖性抑制发生了改变。隐性的pyrH41等位基因有一个单一的C - G到A - T的颠换，将丙氨酸94转化为谷氨酸（A94E）。尽管pyrH41过度表达导致UMP激酶水平远高于野生型菌株，但在这些条件下，carAB操纵子的嘧啶特异性抑制并未恢复。同样，在pyrH41突变体中过度表达盘基网柄菌的UMP - CMP激酶基因，尽管在这种转化体中测得的UMP激酶活性升高，但并未恢复嘧啶介导的carP1启动子活性调控。这些结果表明，除了在嘧啶从头生物合成中的催化功能外，大肠杆菌UMP激酶在carAB操纵子的调控中还发挥了额外的、但以前未被认识到的作用。UMP激酶可能作为内部嘧啶核苷酸库的真正传感器，并与整合宿主因子（IHF）和氨肽酶A（PepA别名CarP和XerB）协同作用，形成carP1启动子活性嘧啶特异性抑制所需的复杂核蛋白结构。
