Studies on stringent control in a cell-free system. Regulation by guanosine-5'-diphosphate-3'-diphosphate of the...

The biosynthesis of elongation factor Tu (EF-Tu) has been studied in a cell-free system with DNA of the transducing phage lambdarifd18 as a template. It was found that the synthesis of EF-Tu in this system was inhibited by about 60% in the presence of 0.3 to 0.6 mM guanosine-5'-diphosphate-3'-diphosphate (ppGpp). The syntheses of several ribosomal proteins encoded in this template, i.e. L1, L10, L11, and L7/L12, were also depressed, whereas those of phage lambda proteins were rather enhanced by the addition of ppGpp. By separating the reaction into two steps, i.e., transcription and translation, the effect of ppGpp was shown to occur at the level of transcription. Several analogs, such as guanosine-5'-diphosphate-3'-monophosphate (ppGp) and guanosine-5'-diphosphate (ppG), were without effect. The formation of mRNA for EF-Tu was assessed directly by specific hybridization with pTUA1 DNA carrying tufA gene. The results clearly indicated that the synthesis of tufB . MRNA was severely and selectively inhibited by ppGpp.