Characterization of RNA and DNA synthesis in Escherichia coli strains devoid of ppGpp.
The synthesis rates of DNA, rRNA, bulk mRNA, protein, and RNA polymerase beta- and beta'-subunits were determined as functions of growth rate in a wild-type Escherichia coli strain, which produces guanosine tetraphosphate (ppGpp), and in a delta relA delta spoT mutant which does not produce ppGpp. The rate of stable RNA synthesis per amount of protein depends on three factors: RNA polymerase concentration, RNA polymerase activity, and the distribution of active RNA polymerase between stable and mRNA genes, measured as the stable RNA synthesis rate/total RNA synthesis rate, rs/rt. In the wild-type strain, all three factors increase with growth rate. In the ppGpp-deficient strains, only RNA polymerase synthesis and activity, but not rs/rt, increased with growth rate. Thus, adjustments of rs/rt require ppGpp. In the absence of ppGpp, the synthesis of rRNA and bulk mRNA both varied in direct proportion to the concentration of active RNA polymerase, in contrast to the wild-type strain, in which only rRNA synthesis increased with growth rate, while mRNA synthesis remained constant. Thus, a control specific for rRNA is absent in strains lacking ppGpp. In rich media, the ppGpp-deficient strain synthesized up to 4-fold more mRNA than wild-type bacteria, which was associated with a similarly increased RNA polymerase activity. We propose that RNA polymerase is rendered inactive in wild-type bacteria due to ppGpp-dependent transcriptional pausing during the synthesis of mRNA. Finally, the control of replication initiation was altered in ppGpp-less bacteria, apparently reflecting indirect changes in the cell physiology, rather than a direct effect of ppGpp on replication initiation.