CONTROL OF THE ESCHERICHIA-COLI RRNB P1 PROMOTER STRENGTH BY PPGPP
CONTROL OF THE ESCHERICHIA-COLI RRNB P1 PROMOTER STRENGTH BY PPGPP
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DOI：
10.1074/jbc.270.19.11181
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发表时间：
1995-05-12
影响因子：
4.8
通讯作者：
BREMER, H
中科院分区：
文献类型：
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作者：
ZHANG, XG;BREMER, H
rrnB P1和P2启动子的融合，和串联P1-P2组合，野生型lacZ基因构建在质粒上，并重组到细菌染色体的mal区域，接近正常位置和rrnB的正常方向。上游激活区（Fis结合位点）始终与P1启动子，所有的构建体包含盒A antitermination rRNA基因位点。使用这些构建体，在大肠杆菌菌株中测量β-半乳糖苷酶的比活性，所述大肠杆菌菌株携带ppGpp合成酶PSI和PSII（relA（+）spoT（+）），或仅携带PSII（Delta relA spoT（+）），或两者都不携带（Delta relA Delta spoT），使用不同的培养基，在37 ℃下支持0.6至2.8道尔顿/小时的生长速率。使用β-半乳糖苷酶活性来估计rrnB P1启动子与分离的rrnB P2启动子相比的相对强度。将启动子强度（每分钟每启动子每游离RNA聚合酶浓度引发的转录物）与启动子活性（每分钟每启动子引发的转录物）区分开。在ppGpp合成（野生型）细菌，rrnB P1启动子的相对强度增加了近10倍，从0.17到1.5的增长率，但在ppGpp少双突变体，它减少了20%，从1.7到1.5。因此，在低水平或零水平的ppGpp下，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的基础水平下强度降低。rrnB P1启动子强度没有额外的ppGpp独立控制是明显的。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的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/分钟增加到33次/分钟，增加了6倍，而分离的P1启动子的活性从2到54起始/分钟增加了24倍。分离的P2启动子的活性增加被认为反映了在恒定启动子强度下RNA聚合酶浓度的增加，而P1启动子活性的急剧增加反映了聚合酶浓度和启动子强度的增加，当与P1串联时，推测P2启动子活性随着P1启动子活性的增加而降低。
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.