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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.
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.
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
6.
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
7.
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
8.
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
9.
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
10.
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.

11.
Mol Microbiol ; 113(6): 1155-1169, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32052499

RESUMO

In bacteria, guanosine (penta)tetra-phosphate ([p]ppGpp) is essential for controlling intracellular metabolism that is needed to adapt to environmental changes, such as amino acid starvation. The (p)ppGpp0 strain of Bacillus subtilis, which lacks (p)ppGpp synthetase, is unable to form colonies on minimal medium. Here, we found suppressor mutations in the (p)ppGpp0 strain, in the purine nucleotide biosynthesis genes, prs, purF and rpoB/C, which encode RNA polymerase core enzymes. In comparing our work with prior studies of ppGpp0 suppressors, we discovered that methionine addition masks the suppression on minimal medium, especially of rpoB/C mutations. Furthermore, methionine addition increases intracellular GTP in rpoB suppressor and this effect is decreased by inhibiting GTP biosynthesis, indicating that methionine addition activated GTP biosynthesis and inhibited growth under amino acid starvation conditions in (p)ppGpp0 backgrounds. Furthermore, we propose that the increase in intracellular GTP levels induced by methionine is due to methionine derivatives that increase the activity of the de novo GTP biosynthesis enzyme, GuaB. Our study sheds light on the potential relationship between GTP homeostasis and methionine metabolism, which may be the key to adapting to environmental changes.


Assuntos
Bacillus subtilis/metabolismo , Guanosina Pentafosfato/metabolismo , Guanosina Trifosfato/biossíntese , Ligases/metabolismo , Metionina/metabolismo , Trifosfato de Adenosina/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Regulação Bacteriana da Expressão Gênica/genética , Ligases/genética , Supressão Genética/genética , Transcrição Genética/genética
12.
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
13.
J Mol Biol ; 431(18): 3568-3590, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-30597160

RESUMO

Within the larger ABC superfamily of ATPases, ABCF family members eEF3 in Saccharomyces cerevisiae and EttA in Escherichia coli have been found to function as ribosomal translation factors. Several other ABCFs including biochemically characterized VgaA, LsaA and MsrE confer resistance to antibiotics that target the peptidyl transferase center and exit tunnel of the ribosome. However, the diversity of ABCF subfamilies, the relationships among subfamilies and the evolution of antibiotic resistance (ARE) factors from other ABCFs have not been explored. To address this, we analyzed the presence of ABCFs and their domain architectures in 4505 genomes across the tree of life. We find 45 distinct subfamilies of ABCFs that are widespread across bacterial and eukaryotic phyla, suggesting that they were present in the last common ancestor of both. Surprisingly, currently known ARE ABCFs are not confined to a distinct lineage of the ABCF family tree, suggesting that ARE can readily evolve from other ABCF functions. Our data suggest that there are a number of previously unidentified ARE ABCFs in antibiotic producers and important human pathogens. We also find that ATPase-deficient mutants of all four E. coli ABCFs (EttA, YbiT, YheS and Uup) inhibit protein synthesis, indicative of their ribosomal function, and demonstrate a genetic interaction of ABCFs Uup and YheS with translational GTPase BipA involved in assembly of the 50S ribosome subunit. Finally, we show that the ribosome-binding resistance factor VmlR from Bacillus subtilis is localized to the cytoplasm, ruling out a role in antibiotic efflux.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Resistência Microbiana a Medicamentos/genética , Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/metabolismo , Transportadores de Cassetes de Ligação de ATP/classificação , Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , Antibacterianos/farmacologia , Bacillus subtilis/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , GTP Fosfo-Hidrolases/metabolismo , Humanos , Modelos Moleculares , Peptidil Transferases/efeitos dos fármacos , Conformação Proteica , Domínios Proteicos , Ribossomos/química , Ribossomos/efeitos dos fármacos , Ribossomos/genética , Saccharomyces cerevisiae/metabolismo
14.
Proc Natl Acad Sci U S A ; 115(36): 8978-8983, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30126986

RESUMO

Many Gram-positive pathogenic bacteria employ ribosomal protection proteins (RPPs) to confer resistance to clinically important antibiotics. In Bacillus subtilis, the RPP VmlR confers resistance to lincomycin (Lnc) and the streptogramin A (SA) antibiotic virginiamycin M (VgM). VmlR is an ATP-binding cassette (ABC) protein of the F type, which, like other antibiotic resistance (ARE) ABCF proteins, is thought to bind to antibiotic-stalled ribosomes and promote dissociation of the drug from its binding site. To investigate the molecular mechanism by which VmlR confers antibiotic resistance, we have determined a cryo-electron microscopy (cryo-EM) structure of an ATPase-deficient B. subtilis VmlR-EQ2 mutant in complex with a B. subtilis ErmDL-stalled ribosomal complex (SRC). The structure reveals that VmlR binds within the E site of the ribosome, with the antibiotic resistance domain (ARD) reaching into the peptidyltransferase center (PTC) of the ribosome and a C-terminal extension (CTE) making contact with the small subunit (SSU). To access the PTC, VmlR induces a conformational change in the P-site tRNA, shifting the acceptor arm out of the PTC and relocating the CCA end of the P-site tRNA toward the A site. Together with microbiological analyses, our study indicates that VmlR allosterically dissociates the drug from its ribosomal binding site and exhibits specificity to dislodge VgM, Lnc, and the pleuromutilin tiamulin (Tia), but not chloramphenicol (Cam), linezolid (Lnz), nor the macrolide erythromycin (Ery).


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Antibacterianos/química , Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Farmacorresistência Bacteriana , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Antibacterianos/farmacologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/química , Ribossomos/genética , Ribossomos/metabolismo
15.
Biosci Biotechnol Biochem ; 82(5): 741-751, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29514560

RESUMO

The WalK/WalR two-component system (TCS), originally identified in Bacillus subtilis, is very highly conserved in gram-positive bacteria, including several important pathogens. The WalK/WalR TCS appears to be involved in the growth of most bacterial species encoding it. Previous studies have indicated conserved functions of this system, defining this signal transduction pathway as a crucial regulatory system for cell wall metabolism. Because of such effects on essential functions, this system is considered a potential target for anti-infective therapeutics. In this review, we discuss the role of WalK/WalR TCS in different bacterial cells, focusing on the function of the genes in its regulon as well as the variations in walRK operon structure, its auxiliary proteins, and the composition of its regulon. We also discuss recent experimental data addressing its essential function and the potential type of signal being sensed by B. subtilis. This review also focuses on the potential future research.

16.
Microbiology (Reading) ; 164(4): 670-684, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29465029

RESUMO

WalRK is an essential two-component signal transduction system that plays a central role in coordinating cell wall synthesis and cell growth in Bacillus subtilis. However, the physiological role of WalRK and its essentiality for growth have not been elucidated. We investigated the behaviour of WalRK during heat stress and its essentiality for cell proliferation. We determined that the inactivation of the walHI genes which encode the negative modulator of WalK, resulted in growth defects and eventual cell lysis at high temperatures. Screening of suppressor mutations revealed that the inactivation of LytE, an dl-endopeptidase, restored the growth of the ΔwalHI mutant at high temperatures. Suppressor mutations that reduced heat induction arising from the walRK regulon were also mapped to the walK ORF. Therefore, we hypothesized that overactivation of LytE affects the phenotype of the ΔwalHI mutant. This hypothesis was corroborated by the overexpression of the negative regulator of LytE, IseA and PdaC, which rescued the growth of the ΔwalHI mutant at high temperatures. Elucidating the cause of the temperature sensitivity of the ΔwalHI mutant could explain the essentiality of WalRK. We proved that the constitutive expression of lytE or cwlO using a synthetic promoter uncouples these expressions from WalRK, and renders WalRK nonessential in the pdaC and iseA mutant backgrounds. We propose that the essentiality of WalRK is derived from the coordination of cell wall metabolism with cell growth by regulating dl-endopeptidase activity under various growth conditions.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Resposta ao Choque Térmico/genética , Regulon/fisiologia , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/fisiologia , Proteínas de Bactérias/genética , Parede Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Mutação , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Fases de Leitura Aberta/genética , Regiões Promotoras Genéticas , Regulon/genética
17.
Front Microbiol ; 9: 3041, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30619132

RESUMO

Cell-free translation systems based on cellular lysates optimized for in vitro protein synthesis have multiple applications both in basic and applied science, ranging from studies of translational regulation to cell-free production of proteins and ribosome-nascent chain complexes. In order to achieve both high activity and reproducibility in a translation system, it is essential that the ribosomes in the cellular lysate are enzymatically active. Here we demonstrate that genomic disruption of genes encoding ribosome inactivating factors - HPF in Bacillus subtilis and Stm1 in Saccharomyces cerevisiae - robustly improve the activities of bacterial and yeast translation systems. Importantly, the elimination of B. subtilis HPF results in a complete loss of 100S ribosomes, which otherwise interfere with disome-based approaches for preparation of stalled ribosomal complexes for cryo-electron microscopy studies.

18.
Nucleic Acids Res ; 45(20): 11525-11534, 2017 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-29036468

RESUMO

Propagation of genetic information is a fundamental property of living organisms. Escherichia coli has a 4.6 Mb circular chromosome with a replication origin, oriC. While the oriC replication has been reconstituted in vitro more than 30 years ago, continuous repetition of the replication cycle has not yet been achieved. Here, we reconstituted the entire replication cycle with 14 purified enzymes (25 polypeptides) that catalyze initiation at oriC, bidirectional fork progression, Okazaki-fragment maturation and decatenation of the replicated circular products. Because decatenation provides covalently closed supercoiled monomers that are competent for the next round of replication initiation, the replication cycle repeats autonomously and continuously in an isothermal condition. This replication-cycle reaction (RCR) propagates ∼10 kb circular DNA exponentially as intact covalently closed molecules, even from a single DNA molecule, with a doubling time of ∼8 min and extremely high fidelity. Very large DNA up to 0.2 Mb is successfully propagated within 3 h. We further demonstrate a cell-free cloning in which RCR selectively propagates circular molecules constructed by a multi-fragment assembly reaction. Our results define the minimum element necessary for the repetition of the chromosome-replication cycle, and also provide a powerful in vitro tool to generate large circular DNA molecules without relying on conventional biological cloning.


Assuntos
Replicação do DNA/genética , DNA Circular/síntese química , Escherichia coli/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Complexo de Reconhecimento de Origem/genética , Sistema Livre de Células/microbiologia , DNA Bacteriano/biossíntese , DNA Bacteriano/genética , DNA Circular/biossíntese , DNA Circular/genética , Origem de Replicação/genética
19.
Structure ; 25(4): 603-616.e4, 2017 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-28286005

RESUMO

The SMC-ScpAB complex plays a crucial role in chromosome organization and segregation in many bacteria. It is composed of a V-shaped SMC dimer and an ScpAB subcomplex that bridges the two Structural Maintenance of Chromosomes (SMC) head domains. Despite its functional significance, the mechanistic details of SMC-ScpAB remain obscure. Here we provide evidence that ATP-dependent head-head engagement induces a lever movement of the SMC neck region, which might help to separate juxtaposed coiled-coil arms. Binding of the ScpA N-terminal domain (NTD) to the SMC neck region is negatively regulated by the ScpB C-terminal domain. Mutations in the ScpA NTD compromise this regulation and profoundly affect the overall shape of the complex. The SMC hinge domain is structurally relaxed when free from coiled-coil juxtaposition. Taken together, we propose that the structural parts of SMC-ScpAB are subjected to the balance between constraint and relaxation, cooperating to modulate dynamic conformational changes of the whole complex.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Cristalografia por Raios X , Modelos Moleculares , Mutação , Ligação Proteica , Multimerização Proteica
20.
PLoS One ; 11(12): e0163057, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28005933

RESUMO

Escherichia coli contains seven rRNA operons, each consisting of the genes for three rRNAs (16S, 23S and 5S rRNA in this order) and one or two tRNA genes in the spacer between 16S and 23S rRNA genes and one or two tRNA genes in the 3' proximal region. All of these rRNA and tRNA genes are transcribed from two promoters, P1 and P2, into single large precursors that are afterward processed to individual rRNAs and tRNAs by a set of RNases. In the course of Genomic SELEX screening of promoters recognized by RNA polymerase (RNAP) holoenzyme containing RpoD sigma, a strong binding site was identified within 16S rRNA gene in each of all seven rRNA operons. The binding in vitro of RNAP RpoD holoenzyme to an internal promoter, referred to the promoter of riRNA (an internal RNA of the rRNA operon), within each 16S rRNA gene was confirmed by gel shift assay and AFM observation. Using this riRNA promoter within the rrnD operon as a representative, transcription in vitro was detected with use of the purified RpoD holoenzyme, confirming the presence of a constitutive promoter in this region. LacZ reporter assay indicated that this riRNA promoter is functional in vivo. The location of riRNA promoter in vivo as identified using a set of reporter plasmids agrees well with that identified in vitro. Based on transcription profile in vitro and Northern blot analysis in vivo, the majority of transcript initiated from this riRNA promoter was estimated to terminate near the beginning of 23S rRNA gene, indicating that riRNA leads to produce the spacer-coded tRNA. Under starved conditions, transcription of the rRNA operon is markedly repressed to reduce the intracellular level of ribosomes, but the levels of both riRNA and its processed tRNAGlu stayed unaffected, implying that riRNA plays a role in the continued steady-state synthesis of tRNAs from the spacers of rRNA operons. We then propose that the tRNA genes organized within the spacers of rRNA-tRNA composite operons are expressed independent of rRNA synthesis under specific conditions where further synthesis of ribosomes is not needed.


Assuntos
Escherichia coli/genética , RNA Ribossômico/metabolismo , RNA de Transferência/metabolismo , Óperon de RNAr/genética , Sítios de Ligação , Northern Blotting , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/metabolismo , Holoenzimas/genética , Holoenzimas/metabolismo , Microscopia de Força Atômica , Regiões Promotoras Genéticas , RNA Ribossômico/genética , RNA de Transferência/genética , Fator sigma/genética , Fator sigma/metabolismo , Transcrição Genética
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