Bauer B F, Kar E G, Elford R M, Holmes W M
Department of Microbiology, Immunology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298.
Gene. 1988;63(1):123-34. doi: 10.1016/0378-1119(88)90551-3.
The promoter for the leuV tRNA operon of Escherichia coli has been studied. Derivatives of this promoter were examined in vivo, fused to the cat gene or to the lacZ gene. When compared to other promoters, the leuV promoter was found to be at least three times stronger than the tyrT promoter (for the tyrT tRNA operon), or the lac promoter (trp::lac promoter fusion) and as strong as the P1,P2 promoter of the rrnB operon (a ribosomal RNA operon). Deletion analysis revealed that, while removal of sequences downstream from +11 (relative to the transcription start point) did not affect activity, removal of sequences upstream from -39 resulted in a ten-fold reduction in expression. Unlike rRNA operons which also display upstream activation, sequences responsible for this effect in the leuV promoter are separated into two regions, one between -76 and -47, and the other between -45 and -39. DNA fragments carrying the leuV promoter migrate aberrantly on polyacrylamide gels, a phenomenon usually associated with DNA bending. One sequence thought to be involved in bending is a TTTTT run centered around -71. Point mutations engineered at this T5 region resulted in a loss of activation but had no apparent effect on migration rate. Transcription efficiency of promoter derivatives was examined in vitro using supercoiled, relaxed, or linearized plasmids as templates. Upstream activation was observed only when using relaxed templates, although maximum activity was obtained using supercoiled forms. Insertion of the very efficient 16S transcription terminator between the leuV promoter and the cat gene resulted in barely detectable activities, indicating that no antitermination mechanism was present.
对大肠杆菌亮氨酸缬氨酸转运RNA操纵子的启动子进行了研究。在体内对该启动子的衍生物进行了检测，将其与氯霉素乙酰转移酶基因（cat基因）或β-半乳糖苷酶基因（lacZ基因）融合。与其他启动子相比，发现亮氨酸缬氨酸启动子的强度至少是酪氨酸转运RNA操纵子的tyrT启动子或乳糖启动子（色氨酸::乳糖启动子融合体）的三倍，与核糖体RNA操纵子rrnB操纵子的P1、P2启动子强度相当。缺失分析表明，虽然去除+11下游的序列（相对于转录起始点）不影响活性，但去除-39上游的序列会导致表达降低十倍。与也显示上游激活的核糖体RNA操纵子不同，亮氨酸缬氨酸启动子中负责这种效应的序列被分成两个区域，一个在-76和-47之间，另一个在-45和-39之间。携带亮氨酸缬氨酸启动子的DNA片段在聚丙烯酰胺凝胶上迁移异常，这种现象通常与DNA弯曲有关。一个被认为参与弯曲的序列是以-71为中心的一串TTTTT。在这个T5区域设计的点突变导致激活丧失，但对迁移率没有明显影响。使用超螺旋、松弛或线性化质粒作为模板在体外检测启动子衍生物的转录效率。仅在使用松弛模板时观察到上游激活，尽管使用超螺旋形式可获得最大活性。在亮氨酸缬氨酸启动子和cat基因之间插入非常有效的16S转录终止子导致活性几乎检测不到，表明不存在抗终止机制。
