Accumulation and turnover of guanosine tetraphosphate in Escherichia coli

Abstract

A strain of Escherichia coli B possessing a temperature-labile valyl-transfer RNA synthetase (strain NF 342) has been used to study guanosine tetraphosphate metabolism. Culturing this strain at a variety of temperatures intermediate between the permissive and non-permissive (30 to 42 °C) yielded a range of growth rates, presumably as a result of varying degrees of limitation for charged valyl-tRNA. The rate of RNA accumulation and the level of guanosine tetraphosphate were measured at these different growth rates. These two parameters were found to be related to one another in a hyperbolic manner, suggesting inhibition of stable RNA synthesis by ppGpp ‡ the concentration corresponding to half-maximal inhibition was approximately 0.1 to 0.2 mM. We suggest a model whereby the transcription initiation frequency of ribosomal RNA and other stringently regulated genes is dependent upon the concentration of a positive control protein, whose transcription-stimulating activity is modulated by the ppGpp concentration. At intermediate temperatures RNA synthesis and growth continue at a reduced rate and a substantial pool of ppGpp accumulates in the cell. This permits entry of exogenous precursors into the intracellular nucleotide pools and enables a study to be made of the interrelation between GTP and ppGpp. Upon amino-acid starvation ppGpp accumulates rapidly, reaching a maximal concentration after 3 to 5 minutes and then declines slightly to a plateau level which is maintained for at least one hour. When [ 3H]guanosine is added 7 minutes after the onset of valyl-tRNA starvation, the specific activity of ppGpp lags about 3 minutes after that of GTP. The kinetics of labelling with [ 3H]guanosine are consistent with a rather direct precursor-product relationship between GTP and ppGpp. After prolonged limitation for charged valyl-tRNA, the steady-state rate of ppGpp formation is about half the rate of ppGpp accumulation occurring immediately after the onset of starvation. Release of amino-acid limitation increases the rate of ppGpp degradation at least fourfold. From this it is inferred that not only the rate of ppGpp synthesis, but the rate of its degradation also, is subject to regulation, presumably involving translational processes.