Effect of variation of charged and uncharged tRNA(Trp) levels on ppGpp synthesis in Escherichia coli.

We introduced into a stringent Escherichia coli tryptophan auxotroph a plasmid bearing the tRNA(Trp) gene under the control of an inducible promoter. This allows us to manipulate the total concentration of tRNA(Trp) in the cell according to whether and when inducer is added to the culture. We also manipulated the concentration of Trp-tRNA(Trp) in vivo since the strain used bears a mutation in the Trp-tRNA synthetase affecting the Km for tryptophan, such that varying the exogenous concentration of tryptophan led to variation in the level of Trp-tRNA(Trp) in the cell. With this system, we found that the signal eliciting ppGpp synthesis during a stringent response triggered by tryptophan limitation did not depend on the absolute concentration of either charged or uncharged tRNA(Trp) but rather depended on a decline in the ratio of charged/uncharged tRNA(Trp). In addition, we found that the amplitude of the response, once triggered by tryptophan limitation, was determined by the total concentration of tRNA(Trp) present in the cell (which is mostly uncharged at that point in time). However, excess uncharged tRNA(Trp) did not amplify ppGpp synthesis triggered by limitation of a different amino acid. These data provide in vivo support for the in vitro-derived model of ppGpp synthesis on ribosomes.