Dattananda C S, Rajkumari K, Gowrishankar J
Centre for Cellular and Molecular Biology, Hyderabad, India.
J Bacteriol. 1991 Dec;173(23):7481-90. doi: 10.1128/jb.173.23.7481-7490.1991.
Transcription of the proU operon in Escherichia coli is induced several hundredfold upon growth of cells in media of elevated osmolarity. A low-copy-number promoter-cloning plasmid vector, with lacZ as the reporter gene, was used for assaying the osmoresponsive promoter activity of each of various lengths of proU DNA, generated by cloning of discrete restriction fragments and by an exonuclease III-mediated deletion approach. The results indicate that expression of proU in E. coli is directed from two promoters, one (P2) characterized earlier by other workers with the start site of transcription 60 nucleotides upstream of the initiation codon of the first structural gene (proV), and the other (P1) situated 250 nucleotides upstream of proV. Furthermore, a region of DNA within proV was shown to be involved in negative regulation of proU transcription; phage Mu dII1681-generated lac fusions in the early region of proV also exhibited partial derepression of proU regulation, in comparison with fusions further downstream in the operon. Sequences around promoter P1, sequences around P2, and the promoter-downstream negative regulatory element, respectively, conferred approximately 5-, 8-, and 25-fold osmoresponsivity on proU expression. Within the region genetically defined to encode the negative regulatory element, there is a 116-nucleotide stretch that is absolutely conserved between the proU operons of E. coli and Salmonella typhimurium and has the capability of exhibiting alternative secondary structure. Insertion of this region of DNA into each of two different plasmid vectors was associated with a marked reduction in the mean topological linking number in plasmid molecules isolated from cultures grown in high-osmolarity medium. We propose that this region of DNA undergoes reversible transition to an underwound DNA conformation under high-osmolarity growth conditions and that this transition mediates its regulatory effect on proU expression.
在高渗透压培养基中培养大肠杆菌时，proU操纵子的转录会被诱导数百倍。一个以lacZ作为报告基因的低拷贝数启动子克隆质粒载体，被用于检测通过克隆离散的限制片段和通过核酸外切酶III介导的缺失方法产生的各种长度的proU DNA的渗透压响应启动子活性。结果表明，大肠杆菌中proU的表达由两个启动子指导，一个（P2）先前由其他研究人员鉴定，其转录起始位点在第一个结构基因（proV）起始密码子上游60个核苷酸处，另一个（P1）位于proV上游250个核苷酸处。此外，proV内的一个DNA区域被证明参与proU转录的负调控；与操纵子中更下游的融合相比，噬菌体Mu dII1681在proV早期区域产生的lac融合也表现出proU调控的部分去阻遏。启动子P1周围的序列、P2周围的序列以及启动子下游的负调控元件分别赋予proU表达约5倍、8倍和25倍的渗透压响应性。在遗传定义为编码负调控元件的区域内，有一段116个核苷酸的序列在大肠杆菌和鼠伤寒沙门氏菌的proU操纵子之间是绝对保守的，并且具有呈现交替二级结构的能力。将该DNA区域插入两种不同的质粒载体中，与从在高渗透压培养基中生长的培养物中分离的质粒分子的平均拓扑连接数显著减少有关。我们提出，该DNA区域在高渗透压生长条件下经历可逆转变为欠旋DNA构象，并且这种转变介导其对proU表达的调控作用。
