Positive control of expression of the argECBH gene cluster in vitro by guanosine 5'-diphosphate 3'-diphosphate

Abstract

By using a cell-free system derived from Escherichia coli, it was found that guanosine 5'-diphosphate 3'-diphosphate (ppGpp) was a positive effector for expression of both wings of the bidirectionally transcribed argECBH gene cluster. A 7- to 20-fold increase in the synthesis of both argininosuccinase (the argH enzyme) and N-acetylornithinase (the argE enzyme) resulted with added ppGpp (0.2 mM optimum). Synthesis of hybridizable argECBH mRNA was enhanced only 30 to 100% by added ppGpp. Of the various guanosine nucleotides tested, only pppGpp mimicked ppGpp. Added ppGpp had no important effect upon (i) measurable argE or argH enzyme activity, (ii) total protein synthesis in the cell-free system, or (iii) the rate of decay of hybridizable argECBH mRNA. With extracts of an argR+ strain, added ppGpp had no effect on the repression of enzyme or mRNA synthesis by L-arginine. By using a two-stage system in which the bulk of argECBH mRNA was synthesized while protein synthesis was delayed, we showed ppGpp acted at some point during transcription.