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1.
ACS Chem Biol ; 16(9): 1680-1691, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34477366

RESUMO

While alarmone nucleotides guanosine-3',5'-bisdiphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3',5'-bisdiphosphate) and pppApp (adenosine-5'-triphosphate-3'-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the RelA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments. To overcome this limitation, we have established an efficient and straightforward procedure for synthesizing nonhydrolysable (p)ppNuNpp analogues starting from 3'-azido-3'-deoxyribonucleotides as key intermediates. To demonstrate the utility of (p)ppGNpp as a molecular tool, we show that (i) as an HD substrate mimic, ppGNpp competes with ppGpp to inhibit the enzymatic activity of human MESH1 Small Alarmone Hyrolase, SAH; and (ii) mimicking the allosteric effects of (p)ppGpp, (p)ppGNpp acts as a positive regulator of the synthetase activity of long ribosome-associated RSHs Rel and RelA. Finally, by solving the structure of the N-terminal domain region (NTD) of T. thermophilus Rel complexed with pppGNpp, we show that as an HD substrate mimic, the analogue serves as a bona fide orthosteric regulator that promotes the same intra-NTD structural rearrangements as the native substrate.

2.
Mol Cell ; 81(16): 3310-3322.e6, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34416138

RESUMO

Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product-the alarmone nucleotide (p)ppGpp-through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function in vivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E. coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity.


Assuntos
Regulação Alostérica/genética , Proteínas de Escherichia coli/genética , GTP Pirofosfoquinase/genética , Guanosina Pentafosfato/genética , Pirofosfatases/genética , Aminoácidos/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Domínio Catalítico/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrolases/genética , Ribossomos/genética , Ribossomos/metabolismo , Inanição/genética , Inanição/metabolismo
3.
Nucleic Acids Res ; 49(14): 8384-8395, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34255843

RESUMO

Bacteria have evolved sophisticated mechanisms to deliver potent toxins into bacterial competitors or into eukaryotic cells in order to destroy rivals and gain access to a specific niche or to hijack essential metabolic or signaling pathways in the host. Delivered effectors carry various activities such as nucleases, phospholipases, peptidoglycan hydrolases, enzymes that deplete the pools of NADH or ATP, compromise the cell division machinery, or the host cell cytoskeleton. Effectors categorized in the family of polymorphic toxins have a modular structure, in which the toxin domain is fused to additional elements acting as cargo to adapt the effector to a specific secretion machinery. Here we show that Photorhabdus laumondii, an entomopathogen species, delivers a polymorphic antibacterial toxin via a type VI secretion system. This toxin inhibits protein synthesis in a NAD+-dependent manner. Using a biotinylated derivative of NAD, we demonstrate that translation is inhibited through ADP-ribosylation of the ribosomal 23S RNA. Mapping of the modification further showed that the adduct locates on helix 44 of the thiostrepton loop located in the GTPase-associated center and decreases the GTPase activity of the EF-G elongation factor.


Assuntos
Toxinas Bacterianas/farmacologia , GTP Fosfo-Hidrolases/genética , RNA Ribossômico 23S/genética , Sistemas de Secreção Tipo VI/efeitos dos fármacos , ADP-Ribosilação/efeitos dos fármacos , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , NAD/genética , Fator G para Elongação de Peptídeos/genética , Photorhabdus/química , Photorhabdus/genética , Biossíntese de Proteínas/efeitos dos fármacos , RNA Ribossômico 23S/efeitos dos fármacos , Tioestreptona/química , Tioestreptona/farmacologia
4.
Mol Cell ; 81(15): 3160-3170.e9, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34174184

RESUMO

RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of small alarmone synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2, and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3' CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some small alarmone hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as RNA-modifying enzymes.


Assuntos
Toxinas Bacterianas/metabolismo , Guanosina Pentafosfato/metabolismo , Ligases/metabolismo , RNA de Transferência/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacologia , Bacilos Gram-Positivos Asporogênicos/química , Bacilos Gram-Positivos Asporogênicos/metabolismo , Guanosina Pentafosfato/química , Ligases/química , Ligases/genética , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/fisiologia , Inibidores da Síntese de Proteínas/farmacologia , Pirofosfatases , Ribossomos/metabolismo
5.
Nucleic Acids Res ; 49(1): 444-457, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33330919

RESUMO

In the Gram-positive Firmicute bacterium Bacillus subtilis, amino acid starvation induces synthesis of the alarmone (p)ppGpp by the RelA/SpoT Homolog factor Rel. This bifunctional enzyme is capable of both synthesizing and hydrolysing (p)ppGpp. To detect amino acid deficiency, Rel monitors the aminoacylation status of the ribosomal A-site tRNA by directly inspecting the tRNA's CCA end. Here we dissect the molecular mechanism of B. subtilis Rel. Off the ribosome, Rel predominantly assumes a 'closed' conformation with dominant (p)ppGpp hydrolysis activity. This state does not specifically select deacylated tRNA since the interaction is only moderately affected by tRNA aminoacylation. Once bound to the vacant ribosomal A-site, Rel assumes an 'open' conformation, which primes its TGS and Helical domains for specific recognition and stabilization of cognate deacylated tRNA on the ribosome. The tRNA locks Rel on the ribosome in a hyperactivated state that processively synthesises (p)ppGpp while the hydrolysis is suppressed. In stark contrast to non-specific tRNA interactions off the ribosome, tRNA-dependent Rel locking on the ribosome and activation of (p)ppGpp synthesis are highly specific and completely abrogated by tRNA aminoacylation. Binding pppGpp to a dedicated allosteric site located in the N-terminal catalytic domain region of the enzyme further enhances its synthetase activity.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Guanosina Pentafosfato/biossíntese , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Acilação , Sítio Alostérico , Bacillus subtilis/genética , Domínio Catalítico , GTP Pirofosfoquinase/metabolismo , Hidrólise , Modelos Genéticos , Modelos Moleculares , Conformação Proteica , Processamento Pós-Transcricional do RNA , Subunidades Ribossômicas Maiores de Bactérias/metabolismo
6.
Front Microbiol ; 11: 277, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32184768

RESUMO

The (p)ppGpp-mediated stringent response is a bacterial stress response implicated in virulence and antibiotic tolerance. Both synthesis and degradation of the (p)ppGpp alarmone nucleotide are mediated by RelA-SpoT Homolog (RSH) enzymes which can be broadly divided in two classes: single-domain 'short' and multi-domain 'long' RSH. The regulatory ACT (Aspartokinase, Chorismate mutase and TyrA)/RRM (RNA Recognition Motif) domain is a near-universal C-terminal domain of long RSHs. Deletion of RRM in both monofunctional (synthesis-only) RelA as well as bifunctional (i.e., capable of both degrading and synthesizing the alarmone) Rel renders the long RSH cytotoxic due to overproduction of (p)ppGpp. To probe the molecular mechanism underlying this effect we characterized Escherichia coli RelA and Bacillus subtilis Rel RSHs lacking RRM. We demonstrate that, first, the cytotoxicity caused by the removal of RRM is counteracted by secondary mutations that disrupt the interaction of the RSH with the starved ribosomal complex - the ultimate inducer of (p)ppGpp production by RelA and Rel - and, second, that the hydrolytic activity of Rel is not abrogated in the truncated mutant. Therefore, we conclude that the overproduction of (p)ppGpp by RSHs lacking the RRM domain is not explained by a lack of auto-inhibition in the absence of RRM or/and a defect in (p)ppGpp hydrolysis. Instead, we argue that it is driven by misregulation of the RSH activation by the ribosome.

7.
Front Microbiol ; 10: 1966, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31507571

RESUMO

Amino acid starvation in Escherichia coli activates the enzymatic activity of the stringent factor RelA, leading to accumulation of the alarmone nucleotide (p)ppGpp. The alarmone acts as an intercellular messenger to regulate transcription, translation and metabolism to mediate bacterial stress adaptation. The enzymatic activity of RelA is subject to multi-layered allosteric control executed both by ligands - such as "starved" ribosomal complexes, deacylated tRNA and pppGpp - and by individual RelA domains. The auto-regulation of RelA is proposed to act either in cis (inhibition of the enzymatic activity of the N-terminal region, NTD, by regulatory C-terminal region, CTD) or in trans (CTD-mediated dimerization leading to enzyme inhibition). In this report, we probed the regulatory roles of the individual domains of E. coli RelA and our results are not indicative of RelA dimerization being the key regulatory mechanism. First, at growth-permitting levels, ectopic expression of RelA CTD does not interfere with activation of native RelA, indicating lack of regulation via inhibitory complex formation in the cell. Second, in our biochemical assays, increasing RelA concentration does not decrease the enzyme activity, as would be expected in the case of efficient auto-inhibition via dimerization. Third, while high-level CTD expression efficiently inhibits the growth, the effect is independent of native RelA and is mediated by direct inhibition of protein synthesis, likely via direct interaction with the ribosomal A-site. Finally, deletion of the RRM domain of the CTD region leads to growth inhibition mediated by accumulation of (p)ppGpp, suggesting de-regulation of the synthetic activity in this mutant.

8.
Mol Microbiol ; 112(4): 1339-1349, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31400173

RESUMO

Bacteria undergoing nutrient starvation induce the ubiquitous stringent response, resulting in gross physiological changes that reprograms cell metabolism from fast to slow growth. The stringent response is mediated by the secondary messengers pppGpp and ppGpp collectively referred to as (p)ppGpp or 'alarmone'. In Escherichia coli, two paralogs, RelA and SpoT, synthesize (p)ppGpp. RelA is activated by amino acid starvation, whereas SpoT, which can also degrade (p)ppGpp, responds to fatty acid (FA), carbon and phosphate starvation. Here, we discover that FA starvation leads to rapid activation of RelA and reveal the underlying mechanism. We show that FA starvation leads to depletion of lysine that, in turn, leads to the accumulation of uncharged tRNALys and activation of RelA. SpoT was also activated by FA starvation but to a lower level and with a delayed kinetics. Next, we discovered that pyruvate, a precursor of lysine, is depleted by FA starvation. We also propose a mechanism that explains how FA starvation leads to pyruvate depletion. Together our results raise the possibility that RelA may be a major player under many starvation conditions previously thought to depend principally on SpoT. Interestingly, FA starvation provoked a ~100-fold increase in relA dependent ampicillin tolerance.


Assuntos
Proteínas de Escherichia coli/metabolismo , Ácidos Graxos/metabolismo , GTP Pirofosfoquinase/metabolismo , Ácido Pirúvico/metabolismo , Aminoácidos/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , GTP Pirofosfoquinase/fisiologia , Regulação Bacteriana da Expressão Gênica/genética , Guanosina Tetrafosfato/metabolismo , Ligases/metabolismo , Lisina/metabolismo , Pirofosfatases/metabolismo , RNA de Transferência/metabolismo
9.
mBio ; 10(3)2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31213559

RESUMO

Type II toxin-antitoxin (TA) modules encode a stable toxin that inhibits cell growth and an unstable protein antitoxin that neutralizes the toxin by direct protein-protein contact. hipBA of Escherichia coli strain K-12 codes for HipA, a serine-threonine kinase that phosphorylates and inhibits glutamyl-tRNA synthetase. Induction of hipA inhibits charging of glutamyl-tRNA that, in turn, inhibits translation and induces RelA-dependent (p)ppGpp synthesis and multidrug tolerance. Here, we describe the discovery of a three-component TA gene family that encodes toxin HipT, which exhibits sequence similarity with the C-terminal part of HipA. A genetic screening revealed that trpS in high copy numbers suppresses HipT-mediated growth inhibition. We show that HipT of E. coli O127 is a kinase that phosphorylates tryptophanyl-tRNA synthetase in vitro at a conserved serine residue. Consistently, induction of hipT inhibits cell growth and stimulates production of (p)ppGpp. The gene immediately upstream from hipT, called hipS, encodes a small protein that exhibits sequence similarity with the N terminus of HipA. HipT kinase was neutralized by cognate HipS in vivo, whereas the third component, HipB, encoded by the first gene of the operon, did not counteract HipT kinase activity. However, HipB augmented the ability of HipS to neutralize HipT. Analysis of two additional hipBST-homologous modules showed that, indeed, HipS functions as an antitoxin in these cases also. Thus, hipBST constitutes a novel family of tricomponent TA modules where hipA has been split into two genes, hipS and hipT, that function as a novel type of TA pair.IMPORTANCE Bacterial toxin-antitoxin (TA) modules confer multidrug tolerance (persistence) that may contribute to the recalcitrance of chronic and recurrent infections. The first high-persister gene identified was hipA of Escherichia coli strain K-12, which encodes a kinase that inhibits glutamyl-tRNA synthetase. The hipA gene encodes the toxin of the hipBA TA module, while hipB encodes an antitoxin that counteracts HipA. Here, we describe a novel, widespread TA gene family, hipBST, that encodes HipT, which exhibits sequence similarity with the C terminus of HipA. HipT is a kinase that phosphorylates tryptophanyl-tRNA synthetase and thereby inhibits translation and induces the stringent response. Thus, this new TA gene family may contribute to the survival and spread of bacterial pathogens.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/enzimologia , Proteínas Serina-Treonina Quinases/genética , Triptofano-tRNA Ligase/antagonistas & inibidores , Toxinas Bacterianas/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Homologia de Sequência de Aminoácidos , Sistemas Toxina-Antitoxina/genética
10.
Sci Rep ; 9(1): 2934, 2019 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-30814571

RESUMO

Cellular growth requires a high level of coordination to ensure that all processes run in concert. The role of the nucleotide alarmone (p)ppGpp has been extensively studied in response to external stresses, such as amino acid starvation, in Escherichia coli, but much less is known about the involvement of (p)ppGpp in response to perturbations in intracellular processes. We therefore employed CRISPRi to transcriptionally repress essential genes involved in 14 vital processes and investigated whether a (p)ppGpp-mediated response would be induced. We show that (p)ppGpp is produced and required for a pertinent stress response during interference with outer membrane biogenesis and ADP synthesis specifically. When these processes were perturbed via the transcriptional repression of essential genes, wild type E. coli MG1655 ceased growing and entered a semi-dormant state, whereas isogenic (p)ppGpp0 cells continued to grow uncontrollably to the point of lysis. Furthermore, in vivo measurements revealed that the ATP levels were intrinsically offset in (p)ppGpp0 cells, further indicating a role for the alarmone in cellular energy homeostasis. In summary, our investigation suggests that (p)ppGpp acts as a coordinator of cell growth in response to imbalances in outer membrane biogenesis and adenosine ribonucleotide synthesis, elucidating novel roles for (p)ppGpp in bacterial physiology.


Assuntos
Metabolismo Energético/fisiologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/metabolismo , Estresse Fisiológico/fisiologia , Trifosfato de Adenosina/metabolismo , Sistemas CRISPR-Cas/genética , Membrana Celular/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Lipopolissacarídeos/biossíntese
11.
Mol Cell ; 70(1): 95-105.e4, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29625042

RESUMO

RelA/SpoT homologs (RSHs) are ubiquitous bacterial enzymes that synthesize and hydrolyze (p)ppGpp in response to environmental challenges. Bacteria cannot survive in hosts and produce infection without activating the (p)ppGpp-mediated stringent response, but it is not yet understood how the enzymatic activities of RSHs are controlled. Using UV crosslinking and deep sequencing, we show that Escherichia coli RelA ((p)ppGpp synthetase I) interacts with uncharged tRNA without being activated. Amino acid starvation leads to loading of cognate tRNA⋅RelA complexes at vacant ribosomal A-sites. In turn, RelA is activated and synthesizes (p)ppGpp. Mutation of a single, conserved residue in RelA simultaneously prevents tRNA binding, ribosome binding, and activation of RelA, showing that all three processes are interdependent. Our results support a model in which (p)ppGpp synthesis occurs by ribosome-bound RelA interacting with the Sarcin-Ricin loop of 23S rRNA.


Assuntos
Escherichia coli K12/enzimologia , Guanosina Tetrafosfato/biossíntese , Ligases/metabolismo , RNA Bacteriano/metabolismo , RNA Ribossômico 23S/metabolismo , RNA de Transferência/metabolismo , Ribossomos/enzimologia , Aminoácidos/deficiência , Sítios de Ligação , Escherichia coli K12/genética , Ligases/genética , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica , RNA Bacteriano/genética , RNA Ribossômico 23S/genética , RNA de Transferência/genética , Ribossomos/genética
12.
J Bacteriol ; 198(14): 1918-1926, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27137501

RESUMO

UNLABELLED: Escherichia coli regulates its metabolism to adapt to changes in the environment, in particular to stressful downshifts in nutrient quality. Such shifts elicit the so-called stringent response, coordinated by the alarmone guanosine tetra- and pentaphosphate [(p)ppGpp]. On sudden amino acid (aa) starvation, RelA [(p)ppGpp synthetase I] activity is stimulated by binding of uncharged tRNAs to a vacant ribosomal site; the (p)ppGpp level increases dramatically and peaks within the time scale of a few minutes. The decrease of the (p)ppGpp level after the peak is mediated by the decreased production of mRNA by (p)ppGpp-associated transcriptional regulation, which reduces the vacant ribosomal A site and thus constitutes negative feedback to the RelA-dependent (p)ppGpp synthesis. Here we showed that on sudden isoleucine starvation, this peak was higher in an E. coli strain that lacks the 10 known mRNase-encoding toxin-antitoxin (TA) modules present in the wild-type (wt) strain. This observation suggested that toxins are part of the negative-feedback mechanism to control the (p)ppGpp level during the early stringent response. We built a ribosome trafficking model to evaluate the fold increase in RelA activity just after the onset of aa starvation. Combining this with a feedback model between the (p)ppGpp level and the mRNA level, we obtained reasonable fits to the experimental data for both strains. The analysis revealed that toxins are activated rapidly, within a minute after the onset of starvation, reducing the mRNA half-life by ∼30%. IMPORTANCE: The early stringent response elicited by amino acid starvation is controlled by a sharp increase of the cellular (p)ppGpp level. Toxin-antitoxin module-encoded mRNases are activated by (p)ppGpp through enhanced degradation of antitoxins. The present work shows that this activation happens over a very short time scale and that the activated mRNases negatively affect the (p)ppGpp level. The proposed mathematical model of (p)ppGpp regulation through the mRNA level highlights the importance of several feedback loops in early (p)ppGpp regulation.


Assuntos
Toxinas Bacterianas/metabolismo , Escherichia coli/enzimologia , Ribonucleases/metabolismo , Antitoxinas/genética , Antitoxinas/metabolismo , Toxinas Bacterianas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Guanosina Tetrafosfato/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribonucleases/genética
13.
Mol Microbiol ; 100(4): 735-47, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26845750

RESUMO

The enteric gamma-proteobacterium Photorhabdus luminescens kills a wide range of insects, whilst also maintaining a mutualistic relationship with soil nematodes from the family Heterorhabditis. Pathogenicity is associated with bacterial exponential growth, whilst mutualism is associated with post-exponential (stationary) phase. During post-exponential growth, P. luminescens also elaborates an extensive secondary metabolism, including production of bioluminescence, antibiotics and pigment. However, the regulatory network that controls the expression of this secondary metabolism is not well understood. The stringent response is a well-described global regulatory system in bacteria and mediated by the alarmone (p)ppGpp. In this study, we disrupted the genes relA and spoT, encoding the two predicted (p)ppGpp synthases of P. luminescens TTO1, and we showed that (p)ppGpp is required for secondary metabolism. Moreover, we found the (p)ppGpp is not required for pathogenicity of P. luminescens, but is required for bacterial survival within the insect cadaver. Finally, we showed that (p)ppGpp is required for P. luminescens to support normal nematode growth and development. Therefore, the regulatory network that controls the transition from pathogenicity to mutualism in P. luminescens requires (p)ppGpp. This is the first report outlining a role for (p)ppGpp in controlling the outcome of an interaction between a bacteria and its host.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Guanosina Pentafosfato/metabolismo , Mariposas/microbiologia , Photorhabdus/patogenicidade , Rhabditoidea/microbiologia , Simbiose , Animais , Antibacterianos/biossíntese , Proteínas de Bactérias/metabolismo , Ligases/genética , Mariposas/fisiologia , Photorhabdus/genética , Photorhabdus/crescimento & desenvolvimento , Photorhabdus/metabolismo , Rhabditoidea/crescimento & desenvolvimento , Metabolismo Secundário , Virulência
14.
Proc Natl Acad Sci U S A ; 112(16): 5171-6, 2015 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-25848049

RESUMO

The model organism Escherichia coli codes for at least 11 type II toxin-antitoxin (TA) modules, all implicated in bacterial persistence (multidrug tolerance). Ten of these encode messenger RNA endonucleases (mRNases) inhibiting translation by catalytic degradation of mRNA, and the 11th module, hipBA, encodes HipA (high persister protein A) kinase, which inhibits glutamyl tRNA synthetase (GltX). In turn, inhibition of GltX inhibits translation and induces the stringent response and persistence. Previously, we presented strong support for a model proposing (p)ppGpp (guanosine tetra and penta-phosphate) as the master regulator of persistence. Stochastic variation of [(p)ppGpp] in single cells induced TA-encoded mRNases via a pathway involving polyphosphate and Lon protease. Polyphosphate activated Lon to degrade all known type II antitoxins of E. coli. In turn, the activated mRNases induced persistence and multidrug tolerance. However, even though it was known that activation of HipA stimulated (p)ppGpp synthesis, our model did not explain how hipBA induced persistence. Here we show that, in support of and consistent with our initial model, HipA-induced persistence depends not only on (p)ppGpp but also on the 10 mRNase-encoding TA modules, Lon protease, and polyphosphate. Importantly, observations with single cells convincingly show that the high level of (p)ppGpp caused by activation of HipA does not induce persistence in the absence of TA-encoded mRNases. Thus, slow growth per se does not induce persistence in the absence of TA-encoded toxins, placing these genes as central effectors of bacterial persistence.


Assuntos
Endonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/metabolismo , Alelos , Antitoxinas/metabolismo , Toxinas Bacterianas/metabolismo , Ativação Enzimática , Modelos Moleculares , RNA Mensageiro/metabolismo , Processos Estocásticos , Imagem com Lapso de Tempo
15.
Nucleic Acids Res ; 43(3): 1529-36, 2015 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-25605801

RESUMO

Collisions between paused transcription elongation complexes and replication forks inevitably happen, which may lead to collapse of replication fork and could be detrimental to cells. Bacterial transcription factor DksA and its cofactor alarmone ppGpp were proposed to contribute to prevention of such collisions, although the mechanism of this activity remains elusive. Here we show that DksA/ppGpp do not destabilise transcription elongation complexes or inhibit their backtracking, as was proposed earlier. Instead, we show, both in vitro and in vivo, that DksA/ppGpp increase fidelity of transcription elongation by slowing down misincorporation events. As misincorporation events cause temporary pauses, contribution to fidelity suggests the mechanism by which DksA/ppGpp contribute to prevention of collisions of transcription elongation complexes with replication forks. DksA is only the second known accessory factor, after transcription factor Gre, that increases fidelity of RNA synthesis in bacteria.


Assuntos
Proteínas de Escherichia coli/fisiologia , Escherichia coli/fisiologia , Pirofosfatases/fisiologia , Transcrição Genética/fisiologia
16.
Nucleic Acids Res ; 41(20): 9257-65, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23935117

RESUMO

Transcription elongation consists of repetition of the nucleotide addition cycle: phosphodiester bond formation, translocation and binding of the next nucleotide. Inhibitor of multi-subunit RNA polymerase tagetitoxin (TGT) enigmatically slows down addition of nucleotides in a sequence-dependent manner, only at certain positions of the template. Here, we show that TGT neither affects chemistry of RNA synthesis nor induces backward translocation, nor competes with the nucleoside triphosphate (NTP) in the active center. Instead, TGT increases the stability of the pre-translocated state of elongation complex, thus slowing down addition of the following nucleotide. We show that the extent of inhibition directly depends on the intrinsic stability of the pre-translocated state. The dependence of translocation equilibrium on the transcribed sequence results in a wide distribution (~1-10(3)-fold) of inhibitory effects of TGT at different positions of the template, thus explaining sequence-specificity of TGT action. We provide biochemical evidence that, in pre-translocated state, TGT stabilizes folded conformation of the Trigger Loop, which inhibits forward and backward translocation of the complex. The results suggest that Trigger Loop folding in the pre-translocated state may serve to reduce back-tracking of the elongation complex. Overall, we propose that translocation may be a limiting and highly regulated step of RNA synthesis.


Assuntos
Ácidos Dicarboxílicos/farmacologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Compostos Organofosforados/farmacologia , Elongação da Transcrição Genética/efeitos dos fármacos , RNA Polimerases Dirigidas por DNA/metabolismo , Difosfatos/metabolismo , Fatores de Elongação da Transcrição/metabolismo
17.
Transcription ; 3(3): 115-8, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22771945

RESUMO

The active center of multi-subunit RNA polymerase consists of two modules, the Mg(2+) module, holding the catalytic Mg(2+) ion, and a module made of a flexible domain, the Trigger Loop. Uniquely, the TL module can be substituted by alternative modules, thus changing the catalytic properties of the active center.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Subunidades Proteicas/genética , Domínio Catalítico , Cátions/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Magnésio/metabolismo , Modelos Moleculares , Subunidades Proteicas/metabolismo , Thermus/genética , Fatores de Transcrição , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo
18.
Nucleic Acids Res ; 39(10): 4352-9, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21266474

RESUMO

The highly processive transcription by multi-subunit RNA polymerases (RNAP) can be interrupted by misincorporation or backtracking events that may stall transcription or lead to erroneous transcripts. Backtracked/misincorporated complexes can be resolved via hydrolysis of the transcript. Here, we show that, in response to misincorporation and/or backtracking, the catalytic domain of RNAP active centre, the trigger loop (TL), is substituted by transcription factor Gre. This substitution turns off the intrinsic TL-dependent hydrolytic activity of RNAP active centre, and exchanges it to a far more efficient Gre-dependent mechanism of RNA hydrolysis. Replacement of the TL by Gre factor occurs only in backtracked/misincorporated complexes, and not in correctly elongating complexes. This controlled switching of RNAP activities allows the processivity of elongation to be unaffected by the hydrolytic activity of Gre, while ensuring efficient proofreading of transcription and resolution of backtracked complexes.


Assuntos
RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética , Domínio Catalítico , Hidrólise , Modelos Moleculares
19.
BMC Biol ; 8: 54, 2010 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-20459653

RESUMO

BACKGROUND: Transcription is the first step of gene expression and is characterized by a high fidelity of RNA synthesis. During transcription, the RNA polymerase active centre discriminates against not just non-complementary ribo NTP substrates but also against complementary 2'- and 3'-deoxy NTPs. A flexible domain of the RNA polymerase active centre, the Trigger Loop, was shown to play an important role in this process, but the mechanisms of this participation remained elusive. RESULTS: Here we show that transcription fidelity is achieved through a multi-step process. The initial binding in the active centre is the major discrimination step for some non-complementary substrates, although for the rest of misincorporation events discrimination at this step is very poor. During the second step, non-complementary and 2'-deoxy NTPs are discriminated against based on differences in reaction transition state stabilization and partly in general base catalysis, for correct versus non-correct substrates. This step is determined by two residues of the Trigger Loop that participate in catalysis. In the following step, non-complementary and 2'-deoxy NTPs are actively removed from the active centre through a rearrangement of the Trigger Loop. The only step of discrimination against 3'-deoxy substrates, distinct from the ones above, is based on failure to orient the Trigger Loop catalytic residues in the absence of 3'OH. CONCLUSIONS: We demonstrate that fidelity of transcription by multi-subunit RNA polymerases is achieved through a stepwise process. We show that individual steps contribute differently to discrimination against various erroneous substrates. We define the mechanisms and contributions of each of these steps to the overall fidelity of transcription.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , DNA/metabolismo , Modelos Moleculares , Transcrição Genética/fisiologia , Cinética , Nucleotídeos/metabolismo
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