Activities of constitutive promoters in Escherichia coli 1 1Edited by D. E. Draper

Liang, Sung-Tzu; Bipatnath, Meeshel; Xu, You; Chen, S.-L; Dennis, Patrick P.; Ehrenberg, Måns; Bremer, H. J.

doi:10.1006/jmbi.1999.3056

Cited by 124 publications

(168 citation statements)

References 69 publications

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“…In addition, resistant clones that survive subsequent glyphosate selection should contain a mutant with enough EPSPS catalytic activity in the presence of glyphosate. The low copy number vector pBR322 (Mayer, 1995) as well as the weak and constitutive bla promoter (Liang et al, 1999) were chosen to provide relatively low EPSPS expression, which not only ensures a low selection background of glyphosate but also selects for a mutant enzyme that exhibits relatively high catalytic activity. It is expected that no mutants, such as the frequently reported G101A mutant (Padgette et al, 1991), will occur in our selection conditions due to the significantly decreased catalytic activity, despite low glyphosate affinity.…”

Section: Discussionmentioning

confidence: 99%

Identification of a Glyphosate-Resistant Mutant of Rice 5-Enolpyruvylshikimate 3-Phosphate Synthase Using a Directed Evolution Strategy

Zhou

Wei

et al. 2005

Plant Physiology

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5-Enolpyruvylshikimate 3-phosphate synthase (EPSPS) is a key enzyme in the shikimate pathway and is targeted by the widespectrum herbicide glyphosate. Here, we describe the use of a selection system based on directed evolution to select glyphosate-resistant mutants of EPSPS. Using this system, the rice (Oryza sativa) EPSPS gene, mutagenized by Error-Prone polymerase chain reaction, was introduced into an EPSPS-deficient Escherichia coli strain, AB2829, and transformants were selected on minimal medium by functional complementation. Three mutants with high glyphosate resistance were identified in three independent glyphosate selection experiments. Each mutant contained a C 317 /T transition within the EPSPS coding sequence, causing a change of proline-106 to leucine (P106L) in the protein sequence. Glyphosate resistance assays indicated a 3-fold increase in glyphosate resistance of E. coli expressing the P106L mutant. Affinity of the P106L mutant for glyphosate and phosphoenolpyruvate was decreased about 70-fold and 4.6-fold, respectively, compared to wild-type EPSPS. Analysis based on a kinetic model demonstrates that the P106L mutant has a high glyphosate resistance while retaining relatively high catalytic efficiency at low phosphoenolpyruvate concentrations. A mathematical model derived from the Michaelis-Menten equation was used to characterize the effect of expression level and selection conditions on kinetic (K i and K m ) variation of the mutants. This prediction suggests that the expression level is an important aspect of the selection system. Furthermore, glyphosate resistance of the P106L mutant was confirmed in transgenic tobacco (Nicotiana tabacum), demonstrating the potential for using the P106L mutant in transgenic crops.

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Section: Discussionmentioning

confidence: 99%

Identification of a Glyphosate-Resistant Mutant of Rice 5-Enolpyruvylshikimate 3-Phosphate Synthase Using a Directed Evolution Strategy

Zhou

Wei

et al. 2005

Plant Physiology

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“…rrn P1 Promoters Are Not Saturated for NTPs in Vivo. It has been proposed that rrn P1 promoters are saturated for their initiating NTPs during exponential growth in vivo, rendering regulation by changing ATP and GTP concentrations unlikely (34). However, when ATP concentrations were increased over the wild-type level by growth of a guaB mutant in guanine (Fig.…”

Section: Ntp-sensing Plays a Role In Homeostaticmentioning

confidence: 98%

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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“…Several features implicit in this proposal have been verified: (i) r-protein promoters are constitutive (78); (ii) r-protein promoters are not always saturated with RNA polymerase, so that their activity depends, indeed, on the extent of repression of other genes (78); (iii) specific r-proteins participate in the regulation of ribosome synthesis (63); and (iv) synthesis of rRNA is specifically regulated (see below). However, the constitutivity of r-protein promoters does not imply that r-protein synthesis is unregulated because the mRNAs of r-protein operons contain internal elements that control their elongation, translation, and lifetime (38,39,63,77,81,84,118).…”

Section: Primary Control Of Ribosomal Protein Synthesismentioning

confidence: 99%

Control of rRNA Synthesis in Escherichia coli : a Systems Biology Approach

Dennis

Ehrenberg

Bremer

2004

Microbiol Mol Biol Rev

163

169

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SUMMARY The first part of this review contains an overview of the various contributions and models relating to the control of rRNA synthesis reported over the last 45 years. The second part describes a systems biology approach to identify the factors and effectors that control the interactions between RNA polymerase and rRNA (rrn) promoters of Escherichia coli bacteria during exponential growth in different media. This analysis is based on measurements of absolute rrn promoter activities as transcripts per minute per promoter in bacterial strains either deficient or proficient in the synthesis of the factor Fis and/or the effector ppGpp. These absolute promoter activities are evaluated in terms of rrn promoter strength (V max/Km ) and free RNA polymerase concentrations. Three major conclusions emerge from this evaluation. First, the rrn promoters are not saturated with RNA polymerase. As a consequence, changes in the concentration of free RNA polymerase contribute to changes in rrn promoter activities. Second, rrn P2 promoter strength is not specifically regulated during exponential growth at different rates; its activity changes only when the concentration of free RNA polymerase changes. Third, the effector ppGpp reduces the strength of the rrn P1 promoter both directly and indirectly by reducing synthesis of the stimulating factor Fis. This control of rrn P1 promoter strength forms part of a larger feedback loop that adjusts the synthesis of ribosomes to the availability of amino acids via amino acid-dependent control of ppGpp accumulation.

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Activities of constitutive promoters in Escherichia coli 1 1Edited by D. E. Draper

Cited by 124 publications

References 69 publications

Identification of a Glyphosate-Resistant Mutant of Rice 5-Enolpyruvylshikimate 3-Phosphate Synthase Using a Directed Evolution Strategy

Identification of a Glyphosate-Resistant Mutant of Rice 5-Enolpyruvylshikimate 3-Phosphate Synthase Using a Directed Evolution Strategy

NTP-sensing by rRNA promoters in Escherichia coli is direct

Control of rRNA Synthesis in Escherichia coli : a Systems Biology Approach

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