Mutational analysis of intervening sequences connecting the binding sites for integration host factor, PepA, PurR, and RNA polymerase in the control region of the Eschetichia coli carAB operon, encoding carbamoylphosphate synthase
Mutational analysis of intervening sequences connecting the binding sites for integration host factor, PepA, PurR, and RNA polymerase in the control region of the Eschetichia coli carAB operon, encoding carbamoylphosphate synthase
复制标题
DOI：
10.1128/jb.188.9.3236-3245.2006
复制
发表时间：
2006-05-01
影响因子：
3.2
通讯作者：
Charlier, D
中科院分区：
文献类型：
--
作者：
Devroede, N;Huysveld, N;Charlier, D
编码大肠杆菌独特的氨基甲酰磷酸合酶的carAB操纵子的转录反映了氨基甲酰磷酸在精氨酸和嘧啶核苷酸生物合成中的双重功能。启动子的串联对由多种机制调节，这取决于两种途径的需要和嘧啶/嘌呤核苷酸平衡的维持。在这里，我们专注于接头区域，施加分布的DNA结合蛋白参与嘧啶和嘌呤特异性抑制的上游启动子P1的靶位点。我们在连接整合宿主因子（IHF）、PepA、PurR和RNA聚合酶的结合位点的四个接头中引入了缺失和插入及其组合，并研究了靶点定相和间隔的重要性以及接头核苷酸序列的重要性。这两个PepA结合位点必须正确对齐，并相对于彼此和启动子分开，用于嘧啶和嘌呤介导的抑制。类似地，IHF和PEPA 2位点的定相和间隔受到严格限制，但仅限于嘧啶特异性抑制。IHF靶点甚至被嘌呤介导的调节所抑制。因此，正确定位PepA内的高阶核蛋白复合物是建立嘧啶介导的抑制和嘌呤和嘧啶依赖性调节之间的耦合的先决条件。我们的数据还表明，存在一种新的顺式作用的嘧啶特异性调节靶点位于位置-60。最后，缺乏其控制区的PI衍生物的分析导致了对过量腺嘌呤对PI的影响的重新评估，并揭示了PI不需要UP元件。
Transcription of the carAB operon encoding the unique carbamoylphosphate synthase of Escherichia coli reflects the dual function of carbamoylphosphate in the biosynthesis of arginine and pyrimidine nucleotides. The tandem pair of promoters is regulated by various mechanisms depending on the needs of both pathways and the maintenance of a pyrimidine/purine nucleotide balance. Here we focus on the linker regions that impose the distribution of target sites for DNA-binding proteins involved in pyrimidine- and purine-specific repression of the upstream promoter P1. We introduced deletions and insertions, and combinations thereof, in four linkers connecting the binding sites for integration host factor (IHF), PepA, PurR, and RNA polymerase and studied the importance of phasing and spacing of the targets and the importance of the nucleotide sequence of the linkers. The two PepA binding sites must be properly aligned and separated with respect to each other and to the promoter for both pyrimidine- and purine-mediated repression. Similarly, the phasing and spacing of the IHF and PEPA2 sites are strictly constrained but only for pyrimidine-specific repression. The IHF target is even dispensable for purine-mediated regulation. Thus, a correct localization of PepA within the higher-order nucleoprotein complex is a prerequisite for the establishment of pyrimidine-mediated repression and for the coupling between purine- and pyrimidine-dependent regulation. Our data also suggest the existence of a novel cis-acting pyrimidine-specific regulatory target located around position -60. Finally, the analysis of a PI derivative devoid of its control region has led to a reappraisal of the effect of excess adenine on PI and has revealed that PI has no need for a UP element.