Isolation and Properties of a Ribosome-bound Factor Required for ppGpp and pppGpp Synthesis in Escherichia coli

A scheme for isolating the stringent factor, a factor which stimulates the pyrophosphorylation of GTP and GDP, from high salt extracts of ribosomes derived from stringent strains of Escherichia coli is reported. The stringent factor was purified 400-fold from the high salt-ribosome eluates. The final preparation was judged to be 85% homogeneous by sedimentation and electrophoretic criteria. The results of sodium dodecyl sulfate-gel ionophoresis and sedimentation analysis indicated that the factor is a protein consisting of a single polypeptide chain with a molecular weight of 77,000. In addition to the stringent factor, the conversion of GTP to the guanosine polyphosphates, ppGpp and pppGpp, was shown to require ATP, Mg2+, and ribosomes. The in vitro reaction was further characterized with respect to kinetic parameters, substrate specificity, and its sensitivity to known inhibitors of the in vivo synthesis of ppGpp. From measurements of stringent factor activity in fractions obtained by differential centrifugation of crude cell extracts it was concluded that essentially all of the factor in the extracts was bound to ribosomes. Extracts prepared from several different relaxed mutants were shown to be devoid of stringent factor activity. Evidence is presented that it is the stringent factor rather than the ribosomes which is impaired in these mutants. The implication of these results in relation to the function of the rel gene and the control mechanism underlying the stringent response is discussed.