Positive control of lac operon expression in vitro by guanosine 5′-diphosphate 3′-diphosphate

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Abstract
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 β-galactosidase (β-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 β-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.