Henrich B, Backes H, Klein J R, Plapp R
Universität Kaiserslautern, Fachbereich Biologie, FRG.
Mol Gen Genet. 1992 Mar;232(1):117-25. doi: 10.1007/BF00299144.
A series of deletions removing progressively larger parts of the 5' flanking region of the Escherichia coli pepD gene was constructed. After fusing the resulting promoter fragments to the chromosomal malPQ operon, their activities were determined by assaying for amylomaltase, the product of the malQ gene. Transcription from the pepD promoter region in exponentially growing cells was estimated to be about 5 times less efficient than transcription from the induced lac promoter. Approximately 115 bp preceding the translation start site of the pepD gene are important for regular promoter functioning, whereas the more distal sequences could be deleted without any significant effects. In bacterial cultures containing limiting amounts of inorganic phosphate, the rate of de novo synthesis of peptidase D, simultaneously with the derepression of alkaline phosphatase, increased about fivefold as a consequence of phosphate starvation. This regulation was shown to occur at the transcriptional level by the use of chromosomal pepD promoter-malPQ fusions. The inducibility by phosphate limitation was conserved in all of the deletion clones in which the pepD promoter region was still functional. As demonstrated by the use of phoB, R, and M mutants, the modulation of pepD expression is independent of the genetic system controlling the pho regulon.
构建了一系列缺失突变体，逐步去除大肠杆菌pepD基因5'侧翼区域越来越大的部分。将所得的启动子片段与染色体上的malPQ操纵子融合后，通过检测malQ基因的产物淀粉麦芽糖酶来确定它们的活性。据估计，在指数生长期细胞中，pepD启动子区域的转录效率比诱导型lac启动子的转录效率低约5倍。pepD基因翻译起始位点之前约115 bp对于启动子的正常功能很重要，而更远端的序列可以被删除而没有任何显著影响。在含有有限量无机磷酸盐的细菌培养物中，由于磷酸盐饥饿，肽酶D的从头合成速率与碱性磷酸酶的去阻遏同时增加了约五倍。通过使用染色体pepD启动子-malPQ融合体表明，这种调节发生在转录水平。在所有pepD启动子区域仍具有功能的缺失克隆中，磷酸盐限制诱导性得以保留。如使用phoB、R和M突变体所证明的，pepD表达的调节独立于控制pho调节子的遗传系统。
