We previously observed that the distribution of RNA polymerase over stable RNA (rRNA, tRNA) and mRNA genes in bacteria depends on the cytoplasmic level of guanosine tetraphosphate (ppGpp). Bacteria have two ppGpp synthetases, PSI and PSII. Whereas PSI is activated during amino acid starvation, PSII synthesizes the basal level of ppGpp during exponential growth. We proposed that the activity of PSII is controlled to adjust ribosome synthesis in different media for the optimization of growth. There have been two major gaps in our knowledge in this regard: the gene for PSII was not known, so that its regulation could not be studied, and the target of ppGpp was not known, so that the mechanism of its action could not be studied. We have now identified the PSII gene, and also isolated mutants that are likely to define the ppGpp target. As next steps, I propose to (1) sequence our PSII mutations and generate a functional map of PSII to aid in the elucidation of its control; (2) find genetic determinants for the control of PSII by manipulating the gene in vitro and studying its effects; (3) map, sequence, and biochemically characterize mutations in the rpoBC region that render rRNA synthesis resistant to high levels of ppGpp; (4) study rrn control under conditions that have only now become available: in the absence of ppGpp in response to different growth media, and in a constant environment in response to induced differences in ppGpp levels.
