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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): 8355-8369, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34255840

RESUMO

In the cell, stalled ribosomes are rescued through ribosome-associated protein quality-control (RQC) pathways. After splitting of the stalled ribosome, a C-terminal polyalanine 'tail' is added to the unfinished polypeptide attached to the tRNA on the 50S ribosomal subunit. In Bacillus subtilis, polyalanine tailing is catalyzed by the NEMF family protein RqcH, in cooperation with RqcP. However, the mechanistic details of this process remain unclear. Here we demonstrate that RqcH is responsible for tRNAAla selection during RQC elongation, whereas RqcP lacks any tRNA specificity. The ribosomal protein uL11 is crucial for RqcH, but not RqcP, recruitment to the 50S subunit, and B. subtilis lacking uL11 are RQC-deficient. Through mutational mapping, we identify critical residues within RqcH and RqcP that are important for interaction with the P-site tRNA and/or the 50S subunit. Additionally, we have reconstituted polyalanine-tailing in vitro and can demonstrate that RqcH and RqcP are necessary and sufficient for processivity in a minimal system. Moreover, the in vitro reconstituted system recapitulates our in vivo findings by reproducing the importance of conserved residues of RqcH and RqcP for functionality. Collectively, our findings provide mechanistic insight into the role of RqcH and RqcP in the bacterial RQC pathway.


Assuntos
Bacillus subtilis/genética , DNA Helicases/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , Peptídeos/genética , Peptídeos/metabolismo , RNA de Transferência , Subunidades Ribossômicas Maiores de Bactérias/genética
4.
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
5.
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
6.
Nat Commun ; 12(1): 3577, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34117249

RESUMO

Target protection proteins confer resistance to the host organism by directly binding to the antibiotic target. One class of such proteins are the antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F-subtype (ARE-ABCFs), which are widely distributed throughout Gram-positive bacteria and bind the ribosome to alleviate translational inhibition from antibiotics that target the large ribosomal subunit. Here, we present single-particle cryo-EM structures of ARE-ABCF-ribosome complexes from three Gram-positive pathogens: Enterococcus faecalis LsaA, Staphylococcus haemolyticus VgaALC and Listeria monocytogenes VgaL. Supported by extensive mutagenesis analysis, these structures enable a general model for antibiotic resistance mediated by these ARE-ABCFs to be proposed. In this model, ABCF binding to the antibiotic-stalled ribosome mediates antibiotic release via mechanistically diverse long-range conformational relays that converge on a few conserved ribosomal RNA nucleotides located at the peptidyltransferase center. These insights are important for the future development of antibiotics that overcome such target protection resistance mechanisms.


Assuntos
Antibacterianos/farmacologia , Diterpenos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Lincosamidas/farmacologia , Compostos Policíclicos/farmacologia , Estreptograminas/farmacologia , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Farmacorresistência Bacteriana/genética , Bactérias Gram-Positivas/genética , Modelos Moleculares , Peptidil Transferases/metabolismo , Conformação Proteica , RNA Mensageiro , Ribossomos/metabolismo
7.
Mol Cell ; 81(1): 115-126.e7, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33259810

RESUMO

In all branches of life, stalled translation intermediates are recognized and processed by ribosome-associated quality control (RQC) pathways. RQC begins with the splitting of stalled ribosomes, leaving an unfinished polypeptide still attached to the large subunit. Ancient and conserved NEMF family RQC proteins target these incomplete proteins for degradation by the addition of C-terminal "tails." How such tailing can occur without the regular suite of translational components is, however, unclear. Using single-particle cryo-electron microscopy (EM) of native complexes, we show that C-terminal tailing in Bacillus subtilis is mediated by NEMF protein RqcH in concert with RqcP, an Hsp15 family protein. Our structures reveal how these factors mediate tRNA movement across the ribosomal 50S subunit to synthesize polypeptides in the absence of mRNA or the small subunit.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/ultraestrutura , Proteínas de Bactérias/genética , Microscopia Crioeletrônica , Subunidades Ribossômicas Maiores de Bactérias/genética , Subunidades Ribossômicas Maiores de Bactérias/ultraestrutura
8.
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
9.
Front Microbiol ; 11: 482585, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33281751

RESUMO

To appropriately switch between sessile and motile lifestyles, bacteria control expression of biofilm-associated genes through multiple regulatory elements. In Pseudomonas aeruginosa, the post-transcriptional regulator RsmA has been implicated in the control of various genes including those related to biofilms, but much of the evidence for these links is limited to transcriptomic and phenotypic studies. RsmA binds to target mRNAs to modulate translation by affecting ribosomal access and/or mRNA stability. Here, we trace a global regulatory role of RsmA to inhibition of the expression of Vfr-a transcription factor that inhibits transcriptional regulator FleQ. FleQ directly controls biofilm-associated genes that encode the PEL polysaccharide biosynthesis machinery. Furthermore, we show that RsmA alone cannot bind vfr mRNA but requires the assistance of RNA chaperone protein Hfq. This is the first example where a RsmA protein family member requires another protein for binding to its target RNA.

10.
Microbiol Mol Biol Rev ; 84(4)2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33177189

RESUMO

Many bacterial pathogens can permanently colonize their host and establish either chronic or recurrent infections that the immune system and antimicrobial therapies fail to eradicate. Antibiotic persisters (persister cells) are believed to be among the factors that make these infections challenging. Persisters are subpopulations of bacteria which survive treatment with bactericidal antibiotics in otherwise antibiotic-sensitive cultures and were extensively studied in a hope to discover the mechanisms that cause treatment failures in chronically infected patients; however, most of these studies were conducted in the test tube. Research into antibiotic persistence has uncovered large intrapopulation heterogeneity of bacterial growth and regrowth but has not identified essential, dedicated molecular mechanisms of antibiotic persistence. Diverse factors and stresses that inhibit bacterial growth reduce killing of the bulk population and may also increase the persister subpopulation, implying that an array of mechanisms are present. Hopefully, further studies under conditions that simulate the key aspects of persistent infections will lead to identifying target mechanisms for effective therapeutic solutions.

11.
Nat Rev Microbiol ; 18(11): 637-648, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32587401

RESUMO

Antibiotic resistance is mediated through several distinct mechanisms, most of which are relatively well understood and the clinical importance of which has long been recognized. Until very recently, neither of these statements was readily applicable to the class of resistance mechanism known as target protection, a phenomenon whereby a resistance protein physically associates with an antibiotic target to rescue it from antibiotic-mediated inhibition. In this Review, we summarize recent progress in understanding the nature and importance of target protection. In particular, we describe the molecular basis of the known target protection systems, emphasizing that target protection does not involve a single, uniform mechanism but is instead brought about in several mechanistically distinct ways.


Assuntos
Antibacterianos , Proteínas de Bactérias , Farmacorresistência Bacteriana/fisiologia , Modelos Moleculares , Antibacterianos/química , Antibacterianos/metabolismo , Bactérias/química , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo
12.
Nat Chem Biol ; 16(8): 834-840, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32393900

RESUMO

Bifunctional Rel stringent factors, the most abundant class of RelA/SpoT homologs, are ribosome-associated enzymes that transfer a pyrophosphate from ATP onto the 3' of guanosine tri-/diphosphate (GTP/GDP) to synthesize the bacterial alarmone (p)ppGpp, and also catalyze the 3' pyrophosphate hydrolysis to degrade it. The regulation of the opposing activities of Rel enzymes is a complex allosteric mechanism that remains an active research topic despite decades of research. We show that a guanine-nucleotide-switch mechanism controls catalysis by Thermus thermophilus Rel (RelTt). The binding of GDP/ATP opens the N-terminal catalytic domains (NTD) of RelTt (RelTtNTD) by stretching apart the two catalytic domains. This activates the synthetase domain and allosterically blocks hydrolysis. Conversely, binding of ppGpp to the hydrolase domain closes the NTD, burying the synthetase active site and precluding the binding of synthesis precursors. This allosteric mechanism is an activity switch that safeguards against futile cycles of alarmone synthesis and degradation.


Assuntos
Proteínas Proto-Oncogênicas c-rel/genética , Proteínas Proto-Oncogênicas c-rel/metabolismo , Sequência de Aminoácidos , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Regulação Bacteriana da Expressão Gênica/genética , Genes rel/genética , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/metabolismo , Hidrolases/metabolismo , Ligases/metabolismo , Ligases/fisiologia , Nucleotídeos/metabolismo , Ribossomos/metabolismo , Thermus thermophilus/enzimologia , Thermus thermophilus/metabolismo
13.
Proc Natl Acad Sci U S A ; 117(19): 10500-10510, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32345719

RESUMO

Under stressful conditions, bacterial RelA-SpoT Homolog (RSH) enzymes synthesize the alarmone (p)ppGpp, a nucleotide second messenger. (p)ppGpp rewires bacterial transcription and metabolism to cope with stress, and, at high concentrations, inhibits the process of protein synthesis and bacterial growth to save and redirect resources until conditions improve. Single-domain small alarmone synthetases (SASs) are RSH family members that contain the (p)ppGpp synthesis (SYNTH) domain, but lack the hydrolysis (HD) domain and regulatory C-terminal domains of the long RSHs such as Rel, RelA, and SpoT. We asked whether analysis of the genomic context of SASs can indicate possible functional roles. Indeed, multiple SAS subfamilies are encoded in widespread conserved bicistronic operon architectures that are reminiscent of those typically seen in toxin-antitoxin (TA) operons. We have validated five of these SASs as being toxic (toxSASs), with neutralization by the protein products of six neighboring antitoxin genes. The toxicity of Cellulomonas marina toxSAS FaRel is mediated by the accumulation of alarmones ppGpp and ppApp, and an associated depletion of cellular guanosine triphosphate and adenosine triphosphate pools, and is counteracted by its HD domain-containing antitoxin. Thus, the ToxSAS-antiToxSAS system with its multiple different antitoxins exemplifies how ancient nucleotide-based signaling mechanisms can be repurposed as TA modules during evolution, potentially multiple times independently.


Assuntos
Bactérias/crescimento & desenvolvimento , Guanosina Pentafosfato/metabolismo , Sistemas Toxina-Antitoxina/fisiologia , Nucleotídeos de Adenina/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Bases de Dados Genéticas , Regulação Bacteriana da Expressão Gênica/genética , Guanosina Tetrafosfato/metabolismo , Guanosina Trifosfato/metabolismo , Ligases/metabolismo , Pirofosfatases/metabolismo , Transdução de Sinais , Estresse Fisiológico/fisiologia
14.
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.

15.
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.

16.
Talanta ; 205: 120161, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31450400

RESUMO

Nucleotides, nucleosides and their derivatives are present in all cells at varying concentrations that change with the nutritional, and energetic status of the cell. Precise measurement of the concentrations of these molecules is instrumental for understanding their regulatory effects. Such measurement is challenging due to the inherent instability of these molecules and, despite many decades of research, the reported values differ widely. Here, we present a comprehensive and easy-to-use approach for determination of the intracellular concentrations of >25 target molecular species. The approach uses rapid filtration and cold acidic extraction followed by high performance liquid chromatography (HPLC) in the hydrophilic interaction liquid chromatography (HILIC) mode using zwitterionic columns coupled with UV and MS detectors. The method reliably detects and quantifies all the analytes expected to be observed in the bacterial cell and paves the way for future studies correlating their concentrations with biological effects.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Escherichia coli K12/isolamento & purificação , Espectrometria de Massas/métodos , Nucleotídeos/química , Concentração de Íons de Hidrogênio , Limite de Detecção , Solventes/química
17.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 8): 561-569, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31397328

RESUMO

The stringent response, controlled by (p)ppGpp, enables bacteria to trigger a strong phenotypic resetting that is crucial to cope with adverse environmental changes and is required for stress survival and virulence. In the bacterial cell, (p)ppGpp levels are regulated by the concerted opposing activities of RSH (RelA/SpoT homologue) enzymes that can transfer a pyrophosphate group of ATP to the 3' position of GDP (or GTP) or remove the 3' pyrophosphate moiety from (p)ppGpp. Bifunctional Rel enzymes are notoriously difficult to crystallize owing to poor stability and a propensity for aggregation, usually leading to a loss of biological activity after purification. Here, the production, biochemical analysis and crystallization of the bifunctional catalytic region of the Rel stringent factor from Thermus thermophilus (RelTtNTD) in the resting state and bound to nucleotides are described. RelTt and RelTtNTD are monomers in solution that are stabilized by the binding of Mn2+ and mellitic acid. RelTtNTD crystallizes in space group P4122, with unit-cell parameters a = b = 88.4, c = 182.7 Å, at 4°C and in space group P41212, with unit-cell parameters a = b = 105.7, c = 241.4 Å, at 20°C.


Assuntos
Proteínas de Bactérias/química , Cristalografia por Raios X/métodos , GTP Pirofosfoquinase/química , Thermus thermophilus/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalização , GTP Pirofosfoquinase/metabolismo , Modelos Moleculares , Conformação Proteica
18.
Nucleic Acids Res ; 47(16): 8807-8820, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31299085

RESUMO

Translation is controlled by numerous accessory proteins and translation factors. In the yeast Saccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related protein, New1, is encoded by a non-essential gene with cold sensitivity and ribosome assembly defect knock-out phenotypes. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is a bona fide assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3. The structure of the ATPase-deficient (EQ2) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 leads to ribosome queuing upstream of 3'-terminal lysine and arginine codons, including those genes encoding proteins of the cytoplasmic translational machinery. Our results suggest that New1 is a translation factor that fine-tunes the efficiency of translation termination or ribosome recycling.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Regulação Fúngica da Expressão Gênica , Terminação Traducional da Cadeia Peptídica , Príons/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Arginina/metabolismo , Sítios de Ligação , Clonagem Molecular , Códon/química , Códon/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Deleção de Genes , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Lisina/metabolismo , Modelos Moleculares , Príons/química , Príons/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
20.
Sci Rep ; 9(1): 3037, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30816176

RESUMO

In addition to the standard set of translation factors common in eukaryotic organisms, protein synthesis in the yeast Saccharomyces cerevisiae requires an ABCF ATPase factor eEF3, eukaryotic Elongation Factor 3. eEF3 is an E-site binder that was originally identified as an essential factor involved in the elongation stage of protein synthesis. Recent biochemical experiments suggest an additional function of eEF3 in ribosome recycling. We have characterised the global effects of eEF3 depletion on translation using ribosome profiling. Depletion of eEF3 results in decreased ribosome density at the stop codon, indicating that ribosome recycling does not become rate limiting when eEF3 levels are low. Consistent with a defect in translation elongation, eEF3 depletion causes a moderate redistribution of ribosomes towards the 5' part of the open reading frames. We observed no E-site codon- or amino acid-specific ribosome stalling upon eEF3 depletion, supporting its role as a general elongation factor. Surprisingly, depletion of eEF3 leads to a relative decrease in P-site proline stalling, which we hypothesise is a secondary effect of generally decreased translation and/or decreased competition for the E-site with eIF5A.


Assuntos
Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Aminoácidos/genética , Sítios de Ligação/genética , Códon de Terminação/genética , Fases de Leitura Aberta/genética , Fatores de Alongamento de Peptídeos/genética , Biossíntese de Proteínas/genética
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