SEQUENCE DETERMINANTS FOR PROMOTER STRENGTH IN THE LEUV OPERON OF ESCHERICHIA-COLI
SEQUENCE DETERMINANTS FOR PROMOTER STRENGTH IN THE LEUV OPERON OF ESCHERICHIA-COLI
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DOI：
10.1016/0378-1119(88)90551-3
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发表时间：
1988-03-15
期刊：
影响因子：
3.5
通讯作者：
HOLMES, WM
中科院分区：
文献类型：
--
作者：
BAUER, BF;KAR, EG;HOLMES, WM
对大肠杆菌亮tRNA操纵子的启动子进行了研究。该启动子的衍生物在体内进行了检查，融合到猫基因或lacZ基因。当与其他启动子相比时，发现leuV启动子比tyrT启动子（对于tyrT tRNA操纵子）或tac启动子（trp：lac启动子融合体）强至少三倍，并且与rrnB操纵子（核糖体RNA操纵子）的P1、P2启动子一样强。缺失分析显示，虽然从+11（相对于转录起始点）下游去除序列不影响活性，但从-39上游去除序列导致表达减少10倍。与也显示上游激活的rRNA操纵子不同，在leuV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leuV启动子的DNA片段在聚丙烯酰胺凝胶上异常迁移，这一现象通常与DNA弯曲有关。被认为涉及弯曲的一个序列是以-71为中心的TTTTT运行。在该T5区域工程化的点突变导致活化丧失，但对迁移率没有明显影响。启动子衍生物的转录效率进行了检查，在体外使用超螺旋，松弛，或线性质粒作为模板。上游激活时，只观察到使用宽松的模板，虽然最大的活动，使用超螺旋形式。在leuV启动子和cat基因之间插入非常有效的16 S转录终止子导致几乎检测不到的活性，表明不存在抗终止机制。
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 tac promoter (trp:lac promoter fusion) and as strong as the P1, P2 promoters 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.