Regulation of Salmonella typhimurium pyr Gene Expression: Effect of Changing Both Purine and Pyrimidine Nucleotide Pools

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...

show abstract

“…2B). These results agree qualitatively with observations reported previously for Salmonella enterica (27).…”

Section: Resultssupporting

confidence: 83%

NTP-sensing by rRNA promoters in Escherichia coli is direct

Schneider

Gaál

Gourse

2002

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

105

View full text Add to dashboard Cite

show abstract

“…This strain exhibits reduced levels of UTP and CTP when cultured with uracil, but the addition of both uracil and cytidine elevates CTP levels (46,47). Growth rates in supplemented MOPS were similar in the presence or absence of cytidine ( ϭ 1.25).…”

Section: Resultsmentioning

confidence: 99%

“…The notion that alterations in the NTP pools could exert control over gene expression was primarily recognized in various nucleotide biosynthetic genes (30,32,45,46,48,49). These studies uncovered a variety of fascinating regulatory strategies resulting from changes in intracellular NTP pools, including alterations in the rate of transcription so as to control the coupling of translation and transcription attenuation (45), alterations in the predominant transcription start site position so as to regulate the synthesis mRNAs with differential capacity to control translation (30,49,50), and stimulation of high rates of reiterative transcription so as to control productive transcript synthesis (39,(51)(52)(53).…”

Section: Discussionmentioning

confidence: 99%

The Escherichia coli fis Promoter Is Regulated by Changes in the Levels of Its Transcription Initiation Nucleotide CTP

Walker

Mallik

Pratt

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

Expression of the Escherichia coli nucleoid-associated protein Fis (factor for inversion stimulation) is controlled at the transcriptional level in accordance with the nutritional availability. It is highly expressed during early logarithmic growth phase in cells growing in rich medium but poorly expressed in late logarithmic and stationary phase. However, fis mRNA expression is prolonged at high levels throughout the logarithmic and early stationary phase when the preferred transcription initiation site (؉1C) is replaced with A or G, indicating that initiation with CTP is a required component of the regulation pattern. We show that RNA polymerase-fis promoter complexes are short lived and that transcription is stimulated over 20-fold from linear or supercoiled DNA if CTP is present during formation of initiation complexes, which serves to stabilize these complexes. Use of fis promoter fusions to lacZ indicated that fis promoter transcription is sensitive to the intracellular pool of the predominant initiating NTP. Growth conditions resulting in increases in CTP pools also result in corresponding increases in fis mRNA levels. Measurements of NTP pools performed throughout the growth of the bacterial culture in rich medium revealed a dramatic increase in all four NTP levels during the transition from stationary to logarithmic growth phase, followed by reproducible oscillations in their levels during logarithmic growth, which later decrease during the transition from logarithmic to stationary phase. In particular, CTP pools fluctuate in a manner consistent with a role in regulating fis expression. These observations support a model whereby fis expression is subject to regulation by the availability of its initiating NTP.Diverse cellular functions have been ascribed to the Escherichia coli Fis (factor for inversion stimulation). In addition to mediating specialized site-specific DNA recombination events (1), this nucleoid-associated protein regulates transcription of ribosomal and tRNAs (2, 3) and a growing number of structural genes (4 -16). Fis levels are transcriptionally regulated according to the nutritional conditions and the growth phase (17-19). A dramatic burst in fis mRNA levels is observed when stationary phase cells are outgrown in rich medium, which reach a peak during early logarithmic growth phase, decrease to much lower levels during late logarithmic growth phase, and become undetectable during stationary phase (17, 18), an expression pattern that is closely followed at the protein level (17,19). The fis mRNA half-life is short (about 2 min) and does not vary appreciably throughout the period of growth phase-dependent expression, indicating that the fis mRNA expression pattern is not attributed to changes in mRNA turnover rates but is primarily attributed to transcriptional control (20). Such drastic changes in intracellular Fis levels are likely to influence its role as a global gene regulator. Prolonged Fis expression well into stationary phase is detrimental to cell viability (21). Hence, cells m...

show abstract

“…Although certain differences in the regulation ofpyrD and pyrC have been noted [8], expression of the two genes parallels one another in most growth conditions in response to fluctuations in the intracellular nucleotide pools [6], and translational attenuation could form a common basis for their regulation.…”

Section: Discussionmentioning

confidence: 99%

Cloning, nucleotide sequence and regulation of the Salmonella typhimurium pyrD gene encoding dihydroorotate dehydrogenase

Frick

Neuhard²,

Kelln

1990

European Journal of Biochemistry

View full text Add to dashboard Cite

The Salmonella typhimurium pyrD gene encoding dihydroorotate dehydrogenase was cloned and sequenced. In total, a sequence of 1286 nucleotide pairs was determined wherein a single open-reading-frame of 1011 bp, encoding a polypeptide of 336 amino acids having 95% similarity with the Escherichia coli pyrD gene product, was identified. A region of hyphenated-dyad symmetry exists within the leader region affording the potential for the formation of a stable secondary structure in the 5' end of the transcript. Mutations from several regulatory mutants were located within the region of dyad symmetry which would impart changes in the transcript within the putative secondary structure, implicating the secondary structure in regulation. Primer extension analysis revealed multiple transcriptional start sites located six to nine nucleotides downstream from the Pribnow box, with the primary initiation site differing in repressing and derepressing growth conditions. The results are discussed in terms of a translational attenuation model for regulation of pyrD expression.In Salmonella typhimurium and Escherichia coli, the pyrD gene encodes dihydroorotate dehydrogenase, the fourth enzyme of the pyrimidine biosynthetic pathway. The enzyme is membrane-bound [l] and linked to the electron transport system of the cell [2]. The E. coli enzyme has been purified and shown to consist of two identical flavin-containing subunits [3, 41. The synthesis of dihydroorotate dehydrogenase is regulated in a complex manner by the intracellular nucleotide pools, being negatively correlated with the concentration of CTP [5, 61 and Each mutation would decrease the stability of the putative secondary structure in the transcript, suggesting that pyrC expression is controlled at the level of translational initiation. A similar involvement of the symmetry regions of the E. coli pyrD and pyrC genes has yet to be demonstrated.To gain further insight into the regulation of pyrD expression in S. typhimuriurn, the gene has been cloned and sequenced. Regulatory mutants overexpressing pyrD have been isolated and the mutations identified. Additionally, the transcriptional start sites in repressing and derepressing conditions have been determined. MATERIALS AND METHODS Bacterial strains, bacteriophages and plasmid vectorsplasmid vectors employed, are listed in Table 1.The S. typhimuriurn LT2 and E. coli K12 strains, and the Media and growth conditionsThe composition of the minimal medium [5] and LB medium [20] has been reported. Glucose and casamino acids were added to minimal medium at 0.2%. Other supplements were added at the following final concentrations: ampicillin (50 pg/ ml) ; tetracycline (10 pg/ml) ; 5-bromo-4-chloro-3-indolyl-~-~-galactopyranoside (40 pg/ml); cytidine (50 pg/ml) ; uracil Regulation was studied in KP1725 which has a mutant pyrH gene encoding a partially defective UMP kinase, and when grown in the absence of exogenous pyrimidines is partially starved for pyrimidine nucleotides. Addition of cytidine and uracil results in swelling of ...

show abstract

Regulation of Salmonella typhimurium pyr Gene Expression: Effect of Changing Both Purine and Pyrimidine Nucleotide Pools

Cited by 22 publications

References 29 publications

NTP-sensing by rRNA promoters in Escherichia coli is direct

NTP-sensing by rRNA promoters in Escherichia coli is direct

The Escherichia coli fis Promoter Is Regulated by Changes in the Levels of Its Transcription Initiation Nucleotide CTP

Cloning, nucleotide sequence and regulation of the Salmonella typhimurium pyrD gene encoding dihydroorotate dehydrogenase

Contact Info

Product

Resources

About