This project involves understanding how changes in Escherichia coli gene expression are coordinated when cell growth is limited by nutrient availability. We are interested in signals provoking changes in cellular levels of regulatory nucleotide analogs of GDP and GTP that bear pyrophosphate residues on the ribose 3' hydroxyl; abbreviated as ppGpp and pppGpp, respectively, or (p)ppGpp collectively. Changes in (p)ppGpp levels are correlated with complex gobal changes in gene expression triggered by sensing nutritional depletion for sources of energy, of amino acids and of phosphate. Multiple sites for regulatory effects of (p)ppGpp probably co-exist at levels of metabolism, transcription and translation. One example in (p)ppGpp induction of expression of the alternative RNA polymerase sigma factor, RpoS, that triggers entry into a globally altered patterns of gene expression characteristic of stationary phase of growth. A 50X induction of RpoS protein occurs without appreciable change in rpoS mRNA levels. In another example, transcription is clearly affected. We reported that mutants of sigma-70, encoded by rpoD, suppress a feature of a (p)ppGpp-deficiency phenotype (multiple amino acid auxotrophy) that is a separate example of global positive regulation by (p)ppGpp. Here regulatory effects are exerted on transcript initiation, judging from altered patterns of abortive RNA chain release in vitro. Last year we noted mutant effects for model promoters with differing abortive RNA chain features; this year we found similar effects for the natural galP2 promoter. We now have verified that the sigma-70 mutants do not alter the kinetic constants for DNA binding or strand separation by RNA polymerase holoenzyme but again diminish RNA chain abortion in vitro while increasing the efficiency of full length transcripts clearly implicating promoter clearance. Resistance to mecillinam, a beta-lactam antibiotic, is a new selectable feature of elevated ppGpp levels; this year we have isolated mecR insertion mutants in relA-deleted hosts revealing several new possible sources of regulation of spoT functions.