Translational control of pyrC expression mediated by nucleotide-sensitive selection of transcriptional start sites in Escherichia coli

Wilson, Helen Rose; Archer, C. D.; Liu, Junke; Turnbough, Charles L.

doi:10.1128/jb.174.2.514-524.1992

Cited by 54 publications

(61 citation statements)

References 30 publications

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“…In addition to these sequence determinants, DNA topology and NTP concentrations also influence TSS selection (6,8,9,11,(21)(22)(23)(24)(25)(26). Thus, TSS selection is a multifactorial process, in which the ultimate outcome for a given promoter reflects the contributions of multiple promoter sequence determinants and multiple reaction conditions.…”

Section: Discussionmentioning

confidence: 99%

Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

Vvedenskaya

Vahedian-Movahed

Zhang

et al. 2016

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

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During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein-DNA interactions with the downstream part of the nontemplate strand of the transcription bubble ("core recognition element," CRE). Here, we investigated whether sequence-specific RNAP-CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP-CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP-CRE interactions on TSS selection in vitro and in vivo for a library of 4 7 (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP-CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5′ merodiploid nativeelongating-transcript sequencing, 5′ mNET-seq, we assessed effects of RNAP-CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP-CRE interactions determine TSS selection. Our findings establish RNAP-CRE interactions are a functional determinant of TSS selection. We propose that RNAP-CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).RNA polymerase | transcription start site selection | promoter | transcription bubble | transcription initiation T ranscription initiation consists of a number of biochemical steps leading to formation of a phosphodiester bond between a nucleoside triphosphate (NTP) bound in the RNA polymerase (RNAP) active-center initiating NTP binding site (i site) and an NTP bound in the RNAP active-center extending NTP binding site (i+1 site) (1-3). For bacterial RNAP, promoter-specific initiation requires the RNAP core enzyme (subunit composition α 2 ββ'ω) to associate with a σ factor forming the RNAP holoenzyme (subunit composition α 2 ββ'ωσ). The σ factor contains determinants for sequence-specific protein-DNA interactions with four core promoter elements: the −35 element, the extended −10 element, the −10 element, and the discriminator element (4).During transcription initiation, RNAP holoenzyme unwinds promoter DNA to form an RNAP-promoter open complex (RPo) containing an unwound, single-stranded "transcription bubble." The process of promoter unwinding begins within the promoter −10 element and propagates downstream, enabling single-stranded nucleotides at the downstream end of the transcription bubble template strand to occup...

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Section: Discussionmentioning

confidence: 99%

Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

Vvedenskaya

Vahedian-Movahed

Zhang

et al. 2016

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

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“…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%

“…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). An effect of the transcription initiation NTP concentration on the kinetics of transcription initiation complex formation was demonstrated in the ribosomal RNA promoter rrnB P1 (54).…”

Section: Discussionmentioning

confidence: 99%

“…␤-Galactosidase assays were performed with cells grown in supplemented C medium, which is NϪ CϪ medium (29) supplemented with 10 mM NH 4 Cl, 0.4 mM MgSO 4 , 0.4% glucose (w/v), 0.2% casamino acids, 1 mM arginine, 20 g/ml tryptophan, 0.015 mM thiamine, and either 1 mM uracil or 0.25 mM UMP (30). A low phosphate growth medium was used to grow cells for 32 P labeling of NTPs.…”

Section: Methodsmentioning

confidence: 99%

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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

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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...

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“…Therefore, we suspect that the mechanism that we have proposed for pyrG regulation in B. subtilis also regulates pyrG expression in other Gram-positive bacteria possessing the conserved sequences. This model for pyrG regulation adds to the rapidly growing list of remarkable mechanisms of gene regulation in which the leader transcript plays a central role in sensing the level of its operon's end-product and then appropriately adjusting the expression of this operon (4,22,(28)(29)(30)(31)(32).…”

Section: Discussionmentioning

confidence: 99%

Attenuation control of pyrG expression in Bacillus subtilis is mediated by CTP-sensitive reiterative transcription

Meng

Turnbough

Switzer

2004

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

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In Bacillus subtilis and other Gram-positive bacteria, pyrimidinemediated regulation of the pyrG gene, which encodes CTP synthetase, occurs through an attenuation mechanism involving an intrinsic transcription terminator in the pyrG leader region. Low intracellular levels of CTP prevent termination at the attenuator by a mechanism that requires the nontemplate strand sequence GGGC at the pyrG transcription initiation site (first G ‫؍‬ ؉1) and the leader transcript sequence GCUCCC located at the 5 end of the terminator RNA hairpin. In this study, we demonstrate that reiterative transcription adds G residues (up to at least 10) to the 5 end of pyrG transcripts when B. subtilis cells are starved for pyrimidines but not when cells are grown with excess cytidine. Regulated repetitive addition of G residues, as well as pyrimidine-mediated pyrG regulation, requires the sequence GGGC or GGGT at the start of pyrG transcription. Mutational insertion of four extra G residues at the 5 end of the pyrG transcript (i.e., 5-GGGGGGGC) results in constitutive pyrG expression. We propose that the incorporation of extra G residues by reiterative transcription at the wild-type promoter occurs when normal transcription elongation is stalled at position ؉4 by low levels of the incoming substrate, CTP, during pyrimidine limitation. The poly(G) extensions on the 5 ends of pyrG transcripts act to prevent transcription attenuation by base pairing with the sequence CUCCCUUUC located in the 5 strand of the terminator hairpin. This control mechanism is likely to operate in other Gram-positive bacteria containing similar pyrG leader sequences.T he bacterial pyrG gene encodes the enzyme CTP synthetase, which aminates UTP to form CTP as the final step of the pyrimidine nucleotide biosynthetic pathway. Recent studies indicate that pyrG expression in the Gram-positive bacteria Bacillus subtilis and Lactococcus lactis is regulated by a CTPsensitive transcription attenuation control mechanism (1-3). In this mechanism, intrinsic transcription termination is controlled at an attenuator immediately preceding the pyrG structural gene, but the means by which the attenuator is regulated by CTP was not determined (2, 3).A clue to the mechanism of pyrG regulation came from a comparison of pyrG leader sequences of several Gram-positive bacteria (2). This comparison showed that the lengths and sequences of the leader regions are not conserved except for three short segments that specify GGGC at the 5Ј end of the pyrG transcript and two complementary sequences, GCUCCC and GGGAGC, which form the base of the stem of the terminator hairpin (Fig. 1). Systematic mutagenesis of the leader region revealed that the conserved sequences specifying GGGC (or a mutant version GGGU) and the hairpin sequence GCUCCC were critical cis-acting elements required for pyrimidinemediated regulation of pyrG expression in B. subtilis (3). Based on these results, it was proposed that transcription termination at the pyrG attenuator was controlled by an RNA-binding protein (3). However...

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Translational control of pyrC expression mediated by nucleotide-sensitive selection of transcriptional start sites in Escherichia coli

Cited by 54 publications

References 30 publications

Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

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

Attenuation control of pyrG expression in Bacillus subtilis is mediated by CTP-sensitive reiterative transcription

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