期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1979
卷号：76
期号：4
页码：1726-1730
DOI：10.1073/pnas.76.4.1726
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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 (lacPrL8UV5), 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 lacPrL8UV5 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.
关键词：coupled protein synthesis in vitro ; transcription initiation ; supercontrol systems ; overlapping metabolic domains ; stringent phenomenon
