Primakoff P, Artz S W
Proc Natl Acad Sci U S A. 1979 Apr;76(4):1726-30. doi: 10.1073/pnas.76.4.1726.
Maximal expression of the Escherichia coli lactose operon in a coupled in vitro transcription-translation system from a Salmonella typhimurium relA mutant was strongly dependent upon addition of guanosine 5'-diphosphate 3'-diphosphate (ppGpp). Without added ppGpp, at saturating 3',5'-cyclic AMP (cAMP) concentrations, synthesis of beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) was reproducibly only 5-7% of that which can be obtained with 0.5-0.8 mM ppGpp. Experiments in which transcription was uncoupled from translation indicated that this 14- to 20-fold stimulation by ppGpp occurred at the level of transcription. When coupled beta-galactosidase synthesis was primed with a template containing a well-characterized mutant lac promoter (lacP(r)L8UV5), the dependence on ppGpp was greatly reduced. This result provides an important experimental control previously unavailable for verifying the significance of ppGpp effects on gene regulation in vitro; it indicates that activation of lacP(+) expression by ppGpp is specifically an effect of increased transcription initiations. Furthermore, the large ppGpp stimulation of lacP(+) DNA enabled the level of expression of this template to approach that of lacP(r)L8UV5 DNA, an observation expected from results in vivo but not obtained with other transcription-translation systems in vitro. The importance of these results is considered with respect to previous ideas on the physiological role of ppGpp as a supercontrol molecule in bacterial regulation.
在来自鼠伤寒沙门氏菌relA突变体的体外转录-翻译偶联系统中，大肠杆菌乳糖操纵子的最大表达强烈依赖于添加鸟苷5'-二磷酸3'-二磷酸（ppGpp）。在不添加ppGpp的情况下，在饱和的3',5'-环腺苷酸（cAMP）浓度下，β-半乳糖苷酶（β-D-半乳糖苷半乳糖水解酶，EC 3.2.1.23）的合成可重复性地仅为在添加0.5 - 0.8 mM ppGpp时所能获得产量的5 - 7%。将转录与翻译解偶联的实验表明，ppGpp的这种14至20倍的刺激作用发生在转录水平。当用含有一个特征明确的突变型乳糖启动子（lacP(r)L8UV5）的模板引发偶联的β-半乳糖苷酶合成时，对ppGpp的依赖性大大降低。这一结果提供了一个重要的实验对照，此前在体外验证ppGpp对基因调控作用的重要性时无法获得；它表明ppGpp对lacP(+)表达的激活具体是转录起始增加的结果。此外，ppGpp对lacP(+) DNA的大幅刺激使得该模板的表达水平接近lacP(r)L8UV5 DNA的表达水平，这一观察结果是体内实验结果所预期的，但在其他体外转录-翻译系统中未得到。本文结合之前关于ppGpp作为细菌调控中的超级控制分子的生理作用的观点，对这些结果的重要性进行了讨论。
