Regulation of rRNA transcription correlates with nucleoside triphosphate sensing.

We have previously shown that the activity of the Escherichia coli rRNA promoter rrnB P1 in vitro depends on the concentration of the initiating nucleotide, ATP, and can respond to changes in ATP pools in vivo. We have proposed that this nucleoside triphosphate (NTP) sensing might contribute to regulation of rRNA transcription. To test this model, we have measured the ATP requirements for transcription from 11 different rrnB P1 core promoter mutants in vitro and compared them with the regulatory responses of the same promoters in vivo. The seven rrnB P1 variants that required much lower ATP concentrations than the wild-type promoter for efficient transcription in vitro were defective for response to growth rate changes in vivo (growth rate-dependent regulation). In contrast, the four variants requiring high ATP concentrations in vitro (like the wild-type promoter) were regulated with the growth rate in vivo. We also observed a correlation between NTP sensing in vitro and the response of the promoters in vivo to deletion of the fis gene (an example of homeostatic control), although this relationship was not as tight as for growth rate-dependent regulation. We conclude that the kinetic features responsible for the high ATP concentration dependence of the rrnB P1 promoter in vitro are responsible, at least in part, for the promoter's regulation in vivo, consistent with the model in which rrnB P1 promoter activity can be regulated by changes in NTP pools in vivo (or by hypothetical factors that work at the same kinetic steps that make the promoter sensitive to NTPs).

Mechanism of regulation of transcription initiation by ppGpp. I. Effects of ppGpp on transcription initiation in vivo and in vitro.

To determine the role of ppGpp in both negative and positive regulation of transcription initiation during exponential growth in Escherichia coli, we examined transcription in vivo and in vitro from the growth-rate-dependent rRNA promoter rrnB P1 and from the inversely growth-rate-dependent amino acid biosynthesis/transport promoters PargI, PhisG, PlysC, PpheA, PthrABC, and PlivJ. rrnB P1 promoter activity was slightly higher at all growth-rates in strains unable to synthesize ppGpp (deltarelAdeltaspoT) than in wild-type strains. Consistent with this observation and with the large decrease in rRNA transcription during the stringent response (when ppGpp levels are much higher), ppGpp inhibited transcription from rrnB P1 in vitro. In contrast, amino acid promoter activity was considerably lower in deltarelAdeltaspoT strains than in wild-type strains, but ppGpp had no effect on amino acid promoter activity in vitro. Detailed kinetic analysis in vitro indicated that open complexes at amino acid promoters formed much more slowly and were much longer-lived than rrnB P1 open complexes. ppGpp did not increase the rates of association with, or escape from, amino acid promoters in vitro, consistent with its failure to stimulate transcription directly. In contrast, ppGpp decreased the half-lives of open complexes at all promoters, whether the half-life was seconds (rrnB P1) or hours (amino acid promoters). The results described here and in the accompanying paper indicate that ppGpp directly inhibits transcription, but only from promoters like rrnB P1 that make short-lived open complexes. The results indicate that stimulation of amino acid promoters occurs indirectly. The accompanying paper evaluates potential models for positive control of amino acid promoters by ppGpp that might explain the requirement of ppGpp for amino acid prototrophy.

Mechanism of regulation of transcription initiation by ppGpp. II. Models for positive control based on properties of RNAP mutants and competition for RNAP.

Strains containing ppGpp, a nucleotide whose synthesis is dependent on the RelA and SpoT proteins of Escherichia coli, display slightly lower rRNA promoter activity and much higher amino acid biosynthesis/transport promoter activity than deltarelAdeltaspoT strains. In the accompanying paper, we show that ppGpp directly inhibits rRNA promoter activity in vitro by decreasing the lifetime of the rrn P1 open complex. However, ppGpp does not stimulate amino acid promoter activity in vitro. We show here that RNA polymerase (RNAP) mutants, selected to confer prototrophy to deltarelAdeltaspoT strains, mimic the effects of ppGpp on wild-type RNAP. Based on the positions of the mutant residues that confer prototrophy in the structure of core RNAP, we suggest molecular models for how the mutants, and by analogy ppGpp, generally decrease the lifetime of open complexes. We show that amino acid promoters require higher concentrations of RNAP for function in vitro and in vivo than control promoters, and are more sensitive to competition for RNAP in vivo than control promoters. Furthermore, we show that the requirement of an amino acid promoter for ppGpp in vivo can be alleviated by increasing its rate-limiting RNAP-binding step. Our data are consistent with a previously proposed passive model in which ppGpp inhibits stable RNA synthesis directly by reducing the lifetime of the rrn P1 open complex, liberating enough RNAP to stimulate transcription from amino acid promoters. Our data also place considerable constraints on models invoking hypothetical factors that might increase amino acid promoter activity in a ppGpp-dependent fashion.