In Vitro Synthesis of ppGpp and pppGpp

Abstract

INTRODUCTION When wild-type or certain strains of E. coli suffer an amino acid “starvation,” they undergo the “stringent” response. The main characteristic of this response is a sharp curtailment in the rate of synthesis of stable RNA species. Mutants that continue to accumulate RNA during amino acid starvation are called relaxed. The classical genetic locus for this effect is the rel A gene. Cashel and Gallant (1969) found that amino acid starvation in stringent strains causes the accumulation of two unusual guanine nucleotides, ppGpp (magic spot I, MSI) and pppGpp (magic spot II, MSII), which did not appear in relaxed strains. They then postulated that high intracellular concentrations of these compounds lead to a cessation of RNA accumulation and to the other physiological changes characteristic of the stringent response. The preceding chapter by Cashel and Gallant reviews the in vivo metabolism of ppGpp and pppGpp and the experiments that implicate these nucleotides as mediators of the stringent response. In vivo experiments strongly suggest that some aspect of the protein synthetic machinery is involved in the synthesis of ppGpp and pppGpp. Cashel and Gallant (1969) speculated that during the stringent response a reaction normally involved in protein synthesis idles and produces ppGpp and pppGpp. This notion is supported by the following evidence: the synthesis of ppGpp and pppGpp during amino acid starvation is dependent upon a high intracellular concentration of uncharged tRNA (Fangman and Neidhardt 1964), the presence of functioning ribosomes (Lund and Kjeldgaard 1972a), and a pool of messenger RNA (Lund...