CARP, INVOLVED IN PYRIMIDINE REGULATION OF THE ESCHERICHIA-COLI CARBAMOYLPHOSPHATE SYNTHETASE OPERON ENCODES A SEQUENCE-SPECIFIC DNA-BINDING PROTEIN IDENTICAL TO XERB AND PEPA, ALSO REQUIRED FOR RESOLUTION OF COLEI MULTIMERS
CARP, INVOLVED IN PYRIMIDINE REGULATION OF THE ESCHERICHIA-COLI CARBAMOYLPHOSPHATE SYNTHETASE OPERON ENCODES A SEQUENCE-SPECIFIC DNA-BINDING PROTEIN IDENTICAL TO XERB AND PEPA, ALSO REQUIRED FOR RESOLUTION OF COLEI MULTIMERS
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DOI：
10.1006/jmbi.1995.0385
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发表时间：
1995-07-21
影响因子：
5.6
通讯作者：
GLANSDORFF, N
中科院分区：
文献类型：
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作者：
CHARLIER, D;HASSANZADEH, G;GLANSDORFF, N
参与大肠杆菌carAB操纵子上游P1启动子的嘧啶特异性调控的carP基因已在体内克隆在微型Mu复制子上，测序并显示与编码氨基肽酶A的xerB（pepA）基因相同，该蛋白也参与Xer介导的ColEI cer位点特异性重组。反式显性等位基因carP 6也被克隆，并显示具有将TGG密码子（Trp 473）转化为TAG琥珀终止密码子的单个G->A转换。缺失31个C-末端氨基酸残基的截短的突变体蛋白显示出部分活性;在多拷贝状态下，carPG等位基因可以恢复carAB启动子P1的嘧啶抑制性。发现单拷贝carPG等位基因的反式显性特征在野生型基因的多个拷贝的存在下被抑制。对carP（pepA）控制区进行测序，并且显示转录起始于三个启动子，其中最上游的一个显示受到负性自动调节。发现CarP（PepA）的氨肽酶活性因其在嘧啶介导的carAB转录抑制中的作用而降低。CarP（PepA）被证明是一种序列特异性DNA结合蛋白，至少在体外不需要任何嘧啶辅因子与DNA结合。迁移率变化和DNA酶I足迹实验表明，纯化的CarP（PepA）与E.大肠杆菌和鼠伤寒沙门氏菌carAB操纵子和一个单一的网站在carP（pepA）控制区。我们建议整合宿主因子和CarP/PepA诱导的carAB控制区的结构修饰引起组装嘧啶特异性核蛋白调控复合物所需的构象变化。
The carP gene involved in pyrimidine-specific regulation of the upstream P1 promoter of the Escherichia coli carAB operon has been cloned in vivo on a mini-Mu replicon, sequenced and shown to be identical to the xerB (pepA) gene encoding aminopeptidase A, a protein also involved in the Xer-mediated site-specific recombination at ColEI cer. The trans-dominant allele carP6 was cloned as well and shown to bear a single G-->A transition that converts the TGG codon (Trp473) into a TAG amber stop codon. The truncated mutant protein, missing the 31 C-terminal amino acid residues, was shown to be partially active; in the multicopy state the carPG allele can restore pyrimidine repressibility of the carAB promoter P1. The trans-dominant character of the single copy carPG allele was found to be suppressed in the presence of multiple copies of the wild-type gene. The carP (pepA) control region was sequenced and transcription shown to be initiated at three promoters, the most upstream one of which was shown to be subject to negative autoregulation. The aminopeptidase activity of CarP (PepA) was found to be dispensable for its role in pyrimidine-mediated repression of carAB transcription. CarP (PepA) was shown to be a sequence-specific DNA-binding protein that does not require, at least not in vitro, any pyrimidine cofactor to bind to the DNA. Mobility-shift and DNase I footprinting experiments have revealed a specific binding of purified CarP (PepA) to two sites in each one of the control regions of the E. coli and Salmonella typhimurium carAB operons and to a single site in the carP (pepA) control region. We propose that integration host factor and CarP/PepA-induced structural modifications in the carAB control region cause conformational changes required to assemble a pyrimidine-specific nucleo-protein regulatory complex.