This is a study of cellular mechanisms that coordinate the changes gene expression during transitions between balanced growth and nutritional impoverishment. Our focus is Escherichia coli and the regulatory roles of analogs of GDP and GTP that bear 3'-pyrophosphate residues, i.e., ppGpp and pppGpp, or (p)ppgpp collectively. Past annual reports have described the genes participating in (p)ppGpp metabolism and their manipulation: the relA gene product is activated to synthesize (p)ppGpp when aminoacyl tRNA is limiting, gppA is responsible for converting pppGpp to ppGpp, and spoT encodes a (p)ppgGpase and an alternate (p)ppGpp synthetic activity. Deletions of both relA and spoT render cells (p)ppGpp deficient and result in a pleiotropic phenotype reflecting the loss of (p)ppGpp-dependent functions, which could be suppressed by mutations mapping in RNA polymerase the rpoB, rpoC, and rpoD subunit genes. This year, the sequence changes of a class of rpoB and all rpoD mutations have been determined. A common basis for the pleiotropic phenotype was established by finding that a single rpoB mutation can be isolated in response to selections that suppress either long term loss of viability or polyaminoacid auxotrophy. This same mutation can confer resistance of rifampicin, lowered transcriptional termination efficiency, and enhanced sensitivity to (p)ppGpp. Sequenced suppressor mutations in rpoD, encoding the sigma-70 subunit responsible for promoter recognition by RNA polymerase, have been found in region 3 which contains a putative helix-turn-helix sequence whose function was previously unknown. Stationary phase-specific gene expression activated by a second sigma-like protein called """"""""sigma-s"""""""" was also discovered as (p)ppGpp-dependent in vivo. Genetic evidence obtained this year may explain the ability of spoT to encode both (p)ppGppase and (p)ppGpp synthesis: the two activities are associated with different polypeptides arising from alternative translational start sites. Also, the identity of the first gene in the spo operon was discovered to be 5'GMP kinase (gmk), another enzyme intimately involved with guanine nucleotide metabolism.
