Cellular mechanisms are studied in Escherichia coli which coordinate the many changes in gene expression that occur during transitions between balanced growth and nutritional impoverishment. We are focused upon regulatory roles played by analogs of GDP and GTP that bear 3'-pyrophosphate residues, abbreviated ppGpp and pppGpp, respectively, or collectively as (p)ppGpp. Past annual reports have described three genes involved in (p)ppGpp metabolism and regulation. 1) The relA gene product is activated to synthesize (p)ppGpp when aminoacylated tRNA is limiting. 2) The spoT gene encodes the major cellular (p)ppGppase, which is inhibited during energy source limitation. Genetic evidence suggests the SpoT protein is also bifunctional and is also required for (p)ppGpp synthesis during energy limitation. 3) The gppA gene product hydrolyzes pppGpp to pGgpp, but not GTP to GDP. Each of these genes has now been exploited to manipulate the total abundance of the regulatory nucleotides or the relative ratios of pppGpp to ppGpp. Earlier, we have made the first eubacterial strains apparently devoid of (p)ppGpp by deleting spoT and relA genes, which results in many unusual effects, including a multiple amino acid auxotrophy. We have mapped mutations reversing these features and found them to occur in genes encoding two subunits of RNA polymerase core enzyme (rpoB and rpoC) and the sigma-70 subunit gene rpoD responsible for promoter recognition. These suppressor mutations appear to limit transcription termination efficiencies and alter the sensitivity of cell growth to (p)ppGpp as well. These properties hint that (p)ppGpp regulatory effects may occur at the level of elongation as well as initiation phases of transcription. A second gene region capable of multicopy complementation of a gppAI missense mutation has been sequenced and surprisingly, found to contain a gene encoding protein sequences resembling arylsulfatases from eukaryotes (humans and sea urchins) as well as a gene with sequence relatedness to an aryisulfatase regulatory protein.
