Swartzman E, Meighen E A
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
J Biol Chem. 1993 Aug 5;268(22):16706-16.
A lux-specific DNA-binding protein was purified to homogeneity from Vibrio harveyi by chromatography on DEAE-Sepharose, DNA-cellulose, Superose 12, and Mono Q. A single polypeptide of M(r) = 23,000 was found on SDS-polyacrylamide gel electrophoresis with an amino-terminal sequence corresponding to that predicted for luxR, a gene that causes a shift in the transcriptional start site from position -123 to -26 base pairs upstream of the initiation codon of luxC in the V. harveyi lux operon and is required for high expression of lux mRNA in recombinant Escherichia coli. Identification of the DNA-binding protein as LuxR was confirmed by showing its absence in V. harveyi luxR-mutants and its synthesis in recombinant E. coli containing V. harveyi luxR. The LuxR protein was shown to bind to two specific (A + T)-rich regions of DNA upstream of the V. harveyi luxC gene: region A, -290 to -253 base pairs, and region B, -170 to -116 base pairs. Synthetic poly(dA-dT) but not poly(dA)-poly(dT) competed with the lux DNA for binding to LuxR suggesting that this protein may be a novel poly(dA-dT)-binding protein in prokaryotes. The LuxR protein inhibited transcription from the -123 promoter in vitro; however, transcription from the -26 promoter was not reconstituted suggesting the possible requirement for other factors in lux gene regulation. LuxR shared sequence identity with two proteins linked to the regulation of enzymes involved in electron transport indicating that it may be a member of a family of regulators of metabolic functions responsible for diverting electrons from the respiratory chain.
通过在DEAE-琼脂糖、DNA-纤维素、Superose 12和Mono Q上进行层析，从哈维弧菌中纯化出了一种与lux特异性结合的DNA结合蛋白，并达到了同质。在SDS-聚丙烯酰胺凝胶电泳上发现了一条分子量为23,000的单一条带，其氨基末端序列与luxR预测的序列相对应，luxR基因可使哈维弧菌lux操纵子中luxC起始密码子上游的转录起始位点从-123位碱基对移至-26位碱基对，并且是重组大肠杆菌中lux mRNA高表达所必需的。通过显示该DNA结合蛋白在哈维弧菌luxR突变体中不存在以及在含有哈维弧菌luxR的重组大肠杆菌中合成，证实了该DNA结合蛋白为LuxR。结果表明，LuxR蛋白可与哈维弧菌luxC基因上游的两个特定富含（A + T）的DNA区域结合：区域A，-290至-253碱基对；区域B，-170至-116碱基对。合成的聚（dA-dT）而非聚（dA）-聚（dT）能与lux DNA竞争结合LuxR，这表明该蛋白可能是原核生物中一种新型的聚（dA-dT）结合蛋白。LuxR蛋白在体外抑制了-123启动子的转录；然而，-26启动子的转录未能重建，这表明lux基因调控可能需要其他因子。LuxR与两种与电子传递相关酶的调控有关的蛋白具有序列同源性，这表明它可能是负责将电子从呼吸链转移的代谢功能调节因子家族的一员。
