Stringent control and growth-rate-dependent control have nonidentical promoter sequence requirements.

Josaitis, Cathleen A.; Gaál, Tamás; Gourse, Richard L.

doi:10.1073/pnas.92.4.1117

Cited by 81 publications

(79 citation statements)

References 36 publications

(41 reference statements)

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“…Although UP elements and Fis sites are required for maximal strength, rrn P1 promoters lacking these sequences (core promoters) are still regulated in response to the cell's nutritional environment (9,10). Consistent with this finding, cells lacking the fis gene regulate transcription from rrn P1 promoters similarly to wild-type strains, because feedback systems compensate for the loss of Fis (7).…”

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confidence: 61%

NTP-sensing by rRNA promoters in Escherichia coli is direct

Schneider

Gaál

Gourse

2002

Proc. Natl. Acad. Sci. U.S.A.

105

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We showed previously that rrn P1 promoters require unusually high concentrations of the initiating nucleoside triphosphates (ATP or GTP, depending on the promoter) for maximal transcription in vitro. We proposed that this requirement for high initiating NTP concentrations contributes to control of the rrn P1 promoters from the seven Escherichia coli rRNA operons. However, the previous studies did not prove that variation in NTP concentration affects rrn P1 promoter activity directly in vivo. Here, we create conditions in vivo in which ATP and GTP concentrations are altered in opposite directions relative to one another, and we show that transcription from rrn P1 promoters that initiate with either ATP or GTP follows the concentration of the initiating NTP for that promoter. These results demonstrate that the effect of initiating NTP concentration on rrn P1 promoter activity in vivo is direct. As predicted by a model in which homeostatic control of rRNA transcription results, at least in part, from sensing of NTP concentrations by rrn P1 promoters, we show that inhibition of protein synthesis results in an increase in ATP concentration and a corresponding increase in transcription from rrnB P1. We conclude that translation is a major consumer of purine NTPs, and that NTP-sensing by rrn P1 promoters serves as a direct regulatory link between translation and ribosome synthesis.B ecause overexpression of ribosomes would be energetically costly, whereas underexpression would prevent the cell from taking full advantage of its nutritional environment, ribosome synthesis is regulated with the demand for protein synthesis. rRNA transcription is the rate-limiting step in ribosome synthesis in Escherichia coli and is controlled by several regulatory mechanisms acting at the level of transcription initiation (1, 2). In addition, an antitermination system ensures efficient rRNA transcription elongation (3).Each of the seven rRNA (rrn) operons in E. coli has two promoters, P1 and P2. The P1 promoters are responsible for the majority of rRNA transcription at moderate to fast growth rates and have been characterized extensively. Much of the intrinsic strength of the rrn P1 promoters results from AϩT-rich sequences (UP elements) upstream of the core promoters that recruit RNA polymerase (RNAP) to the promoter through specific interactions with the RNAP ␣-subunit (4-6). At least two trans-acting proteins affect rRNA transcription. Fis activates transcription from each of the 7 rrn P1 promoters by binding to sites upstream of Ϫ60 relative to the transcription start site, ϩ1 (4, 7), whereas H-NS contributes to repression of rrn P1 promoters during stationary phase (8).Although UP elements and Fis sites are required for maximal strength, rrn P1 promoters lacking these sequences (core promoters) are still regulated in response to the cell's nutritional environment (9, 10). Consistent with this finding, cells lacking the fis gene regulate transcription from rrn P1 promoters similarly to wild-type strains, because feedback systems comp...

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confidence: 61%

NTP-sensing by rRNA promoters in Escherichia coli is direct

Schneider

Gaál

Gourse

2002

Proc. Natl. Acad. Sci. U.S.A.

105

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“…P2 also contains an UP element; however, it has a much smaller effect on promoter activity than that exerted by the P1 UP element (Ross et al, 1993). Both P1 and P2 contain a G+C rich region (Travers, 1984) between the 210 and the transcription start site that is required for stringent control (Josaitis et al, 1995;Murray et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Comparison of Bacteroides thetaiotaomicron and Escherichia coli 16S rRNA gene expression signals

2009

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There are barriers to cross-expression of genes between Bacteroides spp. and Escherichia coli. In this study, a lux-based reporter system was developed for Bacteroides and used to compare the promoter structure and function of a Bacteroides thetaiotaomicron 4001 (BT4001) 16S rRNA promoter with those of E. coli in vivo. Analysis of the BT4001 sequences upstream of the 16S rRNA gene revealed the same overall structure known for E. coli 16S rRNA promoters in that there were two promoters separated by~150 bp. However, the BT4001 16S rRNA promoter contains the proposed Bacteroides "7 and "33 consensus sequences instead of the E. coli "10 and "35 consensus sequences. The biological activity of various configurations of the BT4001 16S rRNA promoter was analysed. Experiments pairing the BT4001 16S rRNA promoter with an E. coli RBS, and vice-versa, confirmed that gene expression between the two species is restricted at the level of transcription. In Bacteroides, a difference in translation initiation also appears to limit expression of foreign genes.

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“…2). Promoters positively regulated by ppGpp have A/T-rich discriminators (DNA sequence between the Ϫ10 hexamer and transcription start site), whereas promoters negatively regulated by ppGpp have G/C-rich discriminators, suggesting that the discriminator sequence is one important feature for regulation by ppGpp (25)(26)(27)(28)(29). Interestingly, the discriminator sequence of the iraP promoter is A/T rich (Fig.…”

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confidence: 98%

ppGpp regulation of RpoS degradation via anti-adaptor protein IraP

Bougdour

Gottesman

2007

Proc. Natl. Acad. Sci. U.S.A.

125

117

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IraP is a small protein that interferes with the delivery of S (RpoS) to the ClpXP protease by blocking the action of RssB, an adaptor protein for S degradation. IraP was previously shown to mediate stabilization of S during phosphate starvation. Here, we show that iraP is transcribed in response to phosphate starvation; this response is mediated by ppGpp. The iraP promoter is positively regulated by ppGpp, dependent on the discriminator region of the iraP promoter. Sensing of phosphate starvation requires SpoT but not RelA. The results demonstrate a target for positive regulation by ppGpp and suggest that the cell use of ppGpp to mediate a variety of starvation responses operates in part by modulating S levels.phosphate starvation ͉ 38 ͉ SpoT B acteria have evolved sophisticated mechanisms to recognize and respond to a variety of environmental stresses. In Escherichia coli, S , the gene product of rpoS, is the factor of the general stress response and adaptation to stationary phase (1, 2). S controls the expression of Ϸ100 genes involved in functions that help cells cope with various kinds of stress. S amounts and activity are tightly regulated (3-5).In exponentially growing cells, the active proteolysis of S is crucial to maintain a low intracellular concentration of this factor (6, 7). S becomes stable as cells are starved or are exposed to certain stresses (reviewed in ref. 4). S protein turnover requires the energy-dependent protease, ClpXP, and the adaptor protein, RssB (SprE), which binds directly to S and targets it to ClpXP (8). RssB has an N-terminal domain of the response regulator family, with a conserved site for phosphorylation. Recently, the identification of an anti-adaptor protein, IraP, provided an explanation for how proteolysis of S can be regulated. In vivo, S is stabilized after phosphate starvation in a manner that depends on IraP and that is independent of RssB phosphorylation. In vitro, IraP inhibits S proteolysis through a direct interaction with RssB (9). Although the inhibitory effect of IraP on S degradation has been observed in response to phosphate starvation, how phosphate starvation is sensed and how this signal affects IraP expression and/or activity has not been described previously. We show in this work that iraP mRNA accumulates in phosphate-starved cells, dependent on the alarmone ppGpp, the global regulator of the stringent response. This induction requires the discriminator sequence of the iraP promoter. Two proteins in E. coli, RelA and SpoT, are capable of synthesizing ppGpp (10, 11); we find that SpoT, but not RelA, is essential for the induction of iraP expression in response to phosphate starvation. The positive regulation of iraP transcription by ppGpp leads to regulation of S proteolysis.Results iraP mRNA Accumulates in Response to Phosphate Starvation. Previous work showed that IraP stabilizes the transcription factor S under phosphate starvation (9). We analyzed by Northern blot the levels of iraP mRNA before and after removal of phosphate. Fig. 1A shows that iraP...

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Stringent control and growth-rate-dependent control have nonidentical promoter sequence requirements.

Cited by 81 publications

References 36 publications

NTP-sensing by rRNA promoters in Escherichia coli is direct

NTP-sensing by rRNA promoters in Escherichia coli is direct

Comparison of Bacteroides thetaiotaomicron and Escherichia coli 16S rRNA gene expression signals

ppGpp regulation of RpoS degradation via anti-adaptor protein IraP

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