Multiple mechanisms are used for growth rate and stringent control of leuVtranscriptional initiation in Escherichia coli

Expression of the Escherichia call leuV operon, which contains three tRNA(1 )(Leu) genes, is regulated by several mechanisms including growth-rate-depe ndent control (GRDC) and stringent control (SC). Structural variants of the leuV promoter which differentially affect these regulatory responses have been identified, suggesting that promoter targets for GRDC and SC may be di fferent and that GRDC of the leuV promoter occurs in the absence of guanosi ne 3',5'-bisdiphosphate. To determine the mechanisms of the leuV promoter r egulation, we have examined the stability of promoter open complexes and th e effects of nucleotide triphosphate (NTP) concentration on the efficiency of the leuV promoter and its structural variants in vitro and in vivo. The leuV promoter open complexes were an order of magnitude more stable to hepa rin challenge than those of runBp(1),. The major initiating nucleotide GTP as well as other NTPs increased the stability of the leuV promoter open com plexes. When the cellular level of purine triphosphates was increased at sl ower growth rates by pyrimidine limitation, a 10% reduction in leuV promote r activity was seen. It therefore appears that transcription initiation fro m the leuV promoter is less sensitive to changes in intracellular NTP conce ntration than that from rrnBp(1),. Comparative analysis of regulation of th e leuV promoter with and without upstream activating sequences (UAS) demons trated that the binding site for factor of inversion stimulation (FIS) loca ted in UAS is essential for maximal GRDC. Moreover, the presence of UAS ove rcame the effects of leuV promoter mutations, which abolished GRDC of the l euV core promoter. However, although the presence of putative FIS binding s ite was essential for optimal GRDC, both mutant and wild-type leuV promoter s containing UAS showed improved GRDC in a fis mutant background, suggestin g that FIS protein is an important but not unique participant in the regula tion of the leuV promoter.