Zhang X, Bremer H
Molecular Program, University of Texas at Dallas, Richardson 75083-0688, USA.
J Biol Chem. 1995 May 12;270(19):11181-9. doi: 10.1074/jbc.270.19.11181.
Fusions of the rrnB P1 and P2 promoters, and of the tandem P1-P2 combination, to a wild-type lacZ gene were constructed on plasmids and recombined into the mal region of the bacterial chromosome, close to the normal location and in the normal orientation of rrnB. The upstream activator region (Fis-binding sites) was always present with the P1 promoter, and all constructs contained the box A antitermination site of rRNA genes. Using these constructs, beta-galactosidase specific activities were measured in Escherichia coli strains carrying either both ppGpp synthetases, PSI and PSII (relA+ spoT+), or only PSII (delta relA spoT+), or neither (delta relA delta spoT), using different media supporting growth rates between 0.6 and 2.8 doublings/h at 37 degrees C. The beta-galactosidase activities were used to estimate the relative strength of the rrnB P1 promoter in comparison to the isolated rrnB P2 promoter. Promoter strength (transcripts initiated per min per promoter per free RNA polymerase concentration) was distinguished from promoter activity (transcripts initiated per min per promoter). In ppGpp-synthesizing (wild-type) bacteria, the relative strength of the rrnB P1 promoter increased nearly 10-fold with increasing growth rate from 0.17 to 1.5, but in the ppGpp-less double mutants it decreased by 20% from 1.7 to 1.5. Thus, at low or zero levels of ppGpp, the P1 promoter was 1.5-1.7 times stronger than the isolated P2 promoter. These results indicate that the normal growth rate control of the rrnB P1 promoter strength requires ppGpp, and that the strength is reduced at basal levels of ppGpp found during exponential growth. No additional ppGpp-independent control of the rrnB P1 promoter strength was evident. From the beta-galactosidase data and previously determined values of rRNA gene activities, the activities of the isolated rrnB P1 and P2 promoters, and of the P2 promoter in the tandem combination, were estimated. With increasing growth rate, the activity of the isolated P2 promoter increased 6-fold from 6 to 33 initiations/min, while the activity of the isolated P1 promoter increased 24-fold from 2 to 54 initiations/min. The increasing activity of the isolated P2 promoter is assumed to reflect the increasing RNA polymerase concentration at constant promoter strength, whereas the steeper increase in P1 promoter activity reflects increases in both polymerase concentration and promoter strength. When in tandem with P1, the P2 promoter activity is inferred to decrease as the P1 promoter activity increases.
将rrnB P1和P2启动子以及串联的P1 - P2组合与野生型lacZ基因融合，构建在质粒上，并重组到细菌染色体的mal区域，靠近rrnB的正常位置且方向正常。上游激活区域（Fis结合位点）总是与P1启动子一起存在，并且所有构建体都包含rRNA基因的A盒抗终止位点。使用这些构建体，在37℃下，在支持生长速率为0.6至2.8代/小时的不同培养基中，测量携带两种ppGpp合成酶PSI和PSII（relA + spoT +）、或仅携带PSII（ΔrelA spoT +）、或两者都不携带（ΔrelA ΔspoT）的大肠杆菌菌株中的β - 半乳糖苷酶比活性。β - 半乳糖苷酶活性用于估计rrnB P1启动子相对于分离的rrnB P2启动子的相对强度。启动子强度（每分钟每个启动子每个游离RNA聚合酶浓度引发的转录本）与启动子活性（每分钟每个启动子引发的转录本）有所区分。在合成ppGpp的（野生型）细菌中，rrnB P1启动子的相对强度随着生长速率从0.17增加到1.5而增加近10倍，但在缺乏ppGpp的双突变体中，它从1.7降至1.5下降了20%。因此，在ppGpp水平较低或为零时，P1启动子比分离的P2启动子强1.5 - 1.7倍。这些结果表明，rrnB P1启动子强度的正常生长速率控制需要ppGpp，并且在指数生长期间发现的ppGpp基础水平下强度会降低。没有明显的额外的不依赖ppGpp的rrnB P1启动子强度控制。根据β - 半乳糖苷酶数据和先前确定的rRNA基因活性值，估计了分离的rrnB P1和P2启动子以及串联组合中P2启动子的活性。随着生长速率的增加，分离的P2启动子的活性从6次起始/分钟增加到33次起始/分钟，增加了6倍，而分离的P1启动子的活性从2次起始/分钟增加到54次起始/分钟，增加了24倍。分离的P2启动子活性的增加被认为反映了在启动子强度恒定的情况下RNA聚合酶浓度的增加，而P1启动子活性更陡峭的增加反映了聚合酶浓度和启动子强度两者的增加。当与P1串联时，推断P2启动子活性随着P1启动子活性的增加而降低。
