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Ribosomal RNA (rRNA) transcription is regulated primarily at the level of initiation from rRNA promoters. The unusual kinetic properties of these promoters result in their specific regulation by two small molecule signals, ppGpp and the initiating NTP, that bind to RNA polymerase (RNAP) at all promoters. We show here that DksA, a protein previously unsuspected as a transcription factor, is absolutely required for rRNA regulation. In deltadksA mutants, rRNA promoters are unresponsive to changes in amino acid availability, growth rate, or growth phase. In vitro, DksA binds to RNAP, reduces open complex lifetime, inhibits rRNA promoter activity, and amplifies effects of ppGpp and the initiating NTP on rRNA transcription, explaining the dksA requirement in vivo. These results expand our molecular understanding of rRNA transcription regulation, may explain previously described pleiotropic effects of dksA, and illustrate how transcription factors that do not bind DNA can nevertheless potentiate RNAP for regulation.

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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).In rapidly dividing Escherichia coli, transcription from the seven rRNA operons accounts for over half of all transcription in the cell (16,26,27,37). rRNA operons are transcribed by two tandem promoters, rrn P1 and rrn P2, with P1 being the predominant promoter at medium to fast growth rates. Several features of rrn P1 promoters contribute to their unusual strength. All seven P1 core promoters contain exact matches to the consensus Ϫ10 hexamer (TATAAT) and close matches to the consensus Ϫ35 hexamer (TTGACA). All of the P1 promoters also derive much of their strength from UP elements, AϩT-rich sequences (located from approximately Ϫ40 to Ϫ60 with respect to the transcription start site) that interact with the ␣ subunit C-terminal domain of RNA polymerase (RNAP) and activate transcription ϳ20-to 50-fold (28, 32, 52). In addition, the transcription factor FIS binds to three to five sites upstream of the UP element in each operon and activates transcription approximately fivefold (32,53).While the rrn P1 promoters can be exceptionally strong, they are also subject to regulatory controls that ensure that energy is not wasted synthesizing excess translation machinery under less favorable growth conditions (16,26,27,37). There are multiple ways in which these control systems have been assayed: responses of the promoters to amino acid starvation (stringent control), to different steady-state growth rates (growth rate-dependent control), and to conditions that e...

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