RESUMO
To assess the influence of degenerate initiation triplets on mRNA recruitment by ribosomes, five mRNAs identical but for their start codon (AUG, GUG, UUG, AUU and AUA) were offered to a limiting amount of ribosomes, alone or in competition with an identical AUGmRNA bearing a mutation conferring different electrophoretic mobility to the product. Translational efficiency and competitiveness of test mRNAs toward this AUGmRNA were determined quantifying the relative amounts of the electrophoretically separated wt and mutated products synthesized in vitro and found to be influenced to different extents by the nature of their initiation triplet and by parameters such as temperature and nutrient availability in the medium. The behaviors of AUAmRNA, UUGmRNA and AUGmRNA were the same between 20 and 40°C whereas the GUG and AUUmRNAs were less active and competed poorly with the AUGmRNA, especially at low temperature. Nutrient limitation and preferential inhibition by ppGpp severely affected activity and competitiveness of all mRNAs bearing non-AUG starts, the UUGmRNA being the least affected. Overall, our data indicate that beyond these effects exclusively due to the degenerate start codons within an optimized translational initiation region, an important role is played by the context in which the rare start codons are present.
Assuntos
Códon de Iniciação , Escherichia coli/genética , Iniciação Traducional da Cadeia Peptídica , Fator de Iniciação 1 em Procariotos/genética , RNA Mensageiro/genética , Ligação Competitiva , Escherichia coli/química , Escherichia coli/metabolismo , Cinética , Mutação , Fator de Iniciação 1 em Procariotos/metabolismo , RNA Mensageiro/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Frações Subcelulares/química , TemperaturaRESUMO
In bacteria, the start site and the reading frame of the messenger RNA are selected by the small ribosomal subunit (30S) when the start codon, typically an AUG, is decoded in the P-site by the initiator tRNA in a process guided and controlled by three initiation factors. This process can be efficiently inhibited by GE81112, a natural tetrapeptide antibiotic that is highly specific toward bacteria. Here GE81112 was used to stabilize the 30S pre-initiation complex and obtain its structure by cryo-electron microscopy. The results obtained reveal the occurrence of changes in both the ribosome conformation and initiator tRNA position that may play a critical role in controlling translational fidelity. Furthermore, the structure highlights similarities with the early steps of initiation in eukaryotes suggesting that shared structural features guide initiation in all kingdoms of life.
Assuntos
Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/genética , RNA de Transferência de Metionina/genética , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Sítios de Ligação , Escherichia coli/genética , Escherichia coli/metabolismo , Células Eucarióticas/metabolismo , Modelos Moleculares , Conformação Molecular , Fatores de Iniciação em Procariotos/química , Fatores de Iniciação em Procariotos/metabolismo , Biossíntese de Proteínas/genética , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA de Transferência de Metionina/química , RNA de Transferência de Metionina/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Subunidades Ribossômicas Menores de Bactérias/químicaRESUMO
GE81112 is a tetrapeptide antibiotic that binds to the 30S ribosomal subunit and specifically inhibits P-site decoding of the mRNA initiation codon by the fMet-tRNA anticodon. GE81112 displays excellent microbiological activity against some Gram-positive and Gram-negative bacteria in both minimal and complete, chemically defined, broth, but is essentially inactive in complete complex media. This is due to the presence of peptides that compete with the antibiotic for the oligopeptide permease system (Opp) responsible for its illicit transport into the bacterial cells as demonstrated in the cases of Escherichia coli and Bacillus subtilis. Mutations that inactivate the Opp system and confer GE81112 resistance arise spontaneously with a frequency of ca. 1 × 10(-6), similar to that of the mutants resistant to tri-l-ornithine, a known Opp substrate. On the contrary, cells expressing extrachromosomal copies of the opp genes are extremely sensitive to GE81112 in rich medium and GE81112-resistant mutations affecting the molecular target of the antibiotic were not detected upon examining >108 cells of this type. However, some mutations introduced in the 16S rRNA to confer kasugamycin resistance were found to reduce the sensitivity of the cells to GE81112.
RESUMO
In prokaryotic systems, the initiation phase of protein synthesis is governed by the presence of initiation factors that guide the transition of the small ribosomal subunit (30S) from an unlocked preinitiation complex (30S preIC) to a locked initiation complex (30SIC) upon the formation of a correct codon-anticodon interaction in the peptidyl (P) site. Biochemical and structural characterization of GE81112, a translational inhibitor specific for the initiation phase, indicates that the main mechanism of action of this antibiotic is to prevent P-site decoding by stabilizing the anticodon stem loop of the initiator tRNA in a distorted conformation. This distortion stalls initiation in the unlocked 30S preIC state characterized by tighter IF3 binding and a reduced association rate for the 50S subunit. At the structural level we observe that in the presence of GE81112 the h44/h45/h24a interface, which is part of the IF3 binding site and forms ribosomal intersubunit bridges, preferentially adopts a disengaged conformation. Accordingly, the findings reveal that the dynamic equilibrium between the disengaged and engaged conformations of the h44/h45/h24a interface regulates the progression of protein synthesis, acting as a molecular switch that senses and couples the 30S P-site decoding step of translation initiation to the transition from an unlocked preIC to a locked 30SIC state.
Assuntos
Antibacterianos/química , Escherichia coli/química , Iniciação Traducional da Cadeia Peptídica , RNA Bacteriano/química , RNA Ribossômico 16S/química , RNA de Transferência/química , Subunidades Ribossômicas Menores de Bactérias/química , Conformação de Ácido NucleicoRESUMO
Hygromycin A (HygA) binds to the large ribosomal subunit and inhibits its peptidyl transferase (PT) activity. The presented structural and biochemical data indicate that HygA does not interfere with the initial binding of aminoacyl-tRNA to the A site, but prevents its subsequent adjustment such that it fails to act as a substrate in the PT reaction. Structurally we demonstrate that HygA binds within the peptidyl transferase center (PTC) and induces a unique conformation. Specifically in its ribosomal binding site HygA would overlap and clash with aminoacyl-A76 ribose moiety and, therefore, its primary mode of action involves sterically restricting access of the incoming aminoacyl-tRNA to the PTC.
Assuntos
Cinamatos/química , Cinamatos/farmacologia , Higromicina B/análogos & derivados , Inibidores da Síntese de Proteínas/química , Inibidores da Síntese de Proteínas/farmacologia , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/efeitos dos fármacos , Sítios de Ligação , Cinamatos/metabolismo , Cristalografia por Raios X , Higromicina B/química , Higromicina B/metabolismo , Higromicina B/farmacologia , Modelos Moleculares , Peptidil Transferases/química , Peptidil Transferases/efeitos dos fármacos , Inibidores da Síntese de Proteínas/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/enzimologia , Subunidades Ribossômicas Maiores de Bactérias/metabolismoRESUMO
A few diatom species produce toxins that affect human and animal health. Among these, members of the Pseudo-nitzschia genus were the first diatoms unambiguously identified as producer of domoic acid, a neurotoxin affecting molluscan shell-fish, birds, marine mammals, and humans. Evidence exists indicating the involvement of another diatom genus, Amphora, as a potential producer of domoic acid. We present a strategy for the detection of the diatom species Amphora coffeaeformis based on the development of species-specific oligonucleotide probes and their application in microarray hybridization experiments. This approach is based on the use of two marker genes highly conserved in all diatoms, but endowed with sufficient genetic divergence to discriminate diatoms at the species level. A region of approximately 450 bp of these previously unexplored marker genes, coding for elongation factor 1-a (eEF1-a) and silicic acid transporter (SIT), was used to design oligonucleotide probes that were tested for specificity in combination with the corresponding fluorescently labeled DNA targets. The results presented in this work suggest a possible use of this DNA chip technology for the selective detection of A. coffeaeformis in environmental settings where the presence of this potential toxin producer may represent a threat to human and animal health. In addition, the same basic approach can be adapted to a wider range of diatoms for the simultaneous detection of microorganisms used as biomarkers of different water quality levels.
Assuntos
Diatomáceas/isolamento & purificação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Itália , Mar Mediterrâneo , Sondas de OligonucleotídeosRESUMO
A chemical derivative of the thiopeptide GE2270A, designated NAI003, was found to possess a substantially reduced antibacterial spectrum in comparison to the parent compound, being active against just a few Gram-positive bacteria. In particular, NAI003 retained low MICs against all tested isolates of Propionibacterium acnes and, to a lesser extent, against Enterococcus faecalis. Furthermore, NAI003 showed a time- and dose-dependent killing of both a clindamycin-resistant and a clindamycin-sensitive P. acnes isolate. Gel shift experiments indicated that, like the parent compound, NAI003 retained the ability to bind to elongation factors Tu (EF-Tus) derived from Escherichia coli, E. faecalis, or P. acnes, albeit with reduced efficiency. In contrast, EF-Tus derived from the NAI003-insensitive Staphylococcus aureus or Streptococcus pyogenes did not bind this compound. These results were confirmed by in vitro studies using a hybrid translation system, which indicated that NAI003 can inhibit most efficiently protein synthesis driven by the P. acnes EF-Tu. P. acnes mutants resistant to NAI003 were isolated by direct plating. With one exception, all analyzed strains carried mutations in the tuf gene, encoding EF-Tu. Because of its selective effect on P. acnes in comparison to resident skin flora, NAI003 represents a promising candidate for the topical treatment of acne, which has already completed a phase 1 clinical study.
Assuntos
Antibacterianos/farmacologia , Peptídeos Cíclicos/farmacologia , Propionibacterium acnes/efeitos dos fármacos , Tiazóis/farmacologia , Administração Tópica , Clindamicina/farmacologia , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Fator Tu de Elongação de Peptídeos/metabolismo , Propionibacterium acnes/metabolismo , Pele/microbiologiaRESUMO
Our understanding of the composition of diatom communities and their response to environmental changes is currently limited by laborious taxonomic identification procedures. Advances in molecular technologies are expected to contribute more efficient, robust and sensitive tools for the detection of these ecologically relevant microorganisms. There is a need to explore and test phylogenetic markers as an alternative to the use of rRNA genes, whose limited sequence divergence does not allow the accurate discrimination of diatoms at the species level. In this work, nine diatom species belonging to eight genera, isolated from epylithic environmental samples collected in central Italy, were chosen to implement a panel of diatoms covering the full range of ecological status of freshwaters. The procedure described in this work relies on the PCR amplification of specific regions in two conserved diatom genes, elongation factor 1-a (eEF1-a) and silicic acid transporter (SIT), as a first step to narrow down the complexity of the targets, followed by microarray hybridization experiments. Oligonucleotide probes with the potential to discriminate closely related species were designed taking into account the genetic polymorphisms found in target genes. These probes were tested, refined and validated on a small-scale prototype DNA chip. Overall, we obtained 17 highly specific probes targeting eEF1-a and SIT, along with 19 probes having lower discriminatory power recognizing at the same time two or three species. This basic array was validated in a laboratory setting and is ready for tests with crude environmental samples eventually to be scaled-up to include a larger panel of diatoms. Its possible use for the simultaneous detection of diatoms selected from the classes of water quality identified by the European Water Framework Directive is discussed.
Assuntos
Biomarcadores/análise , Diatomáceas/isolamento & purificação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fator 1 de Elongação de Peptídeos/genética , Sequência de Bases , Diatomáceas/classificação , Diatomáceas/genética , Água Doce , Itália , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Qualidade da ÁguaRESUMO
The translocation of mRNA and tRNA through the ribosome is catalyzed by elongation factor G (EF-G), a universally conserved guanosine triphosphate hydrolase (GTPase). The mechanism by which the closely related decapeptide antibiotics dityromycin and GE82832 inhibit EF-G-catalyzed translocation is elucidated in this study. Using crystallographic and biochemical experiments, we demonstrate that these antibiotics bind to ribosomal protein S12 in solution alone as well as within the small ribosomal subunit, inducing long-range effects on the ribosomal head. The crystal structure of the antibiotic in complex with the 70S ribosome reveals that the binding involves conserved amino acid residues of S12 whose mutations result in in vitro and in vivo antibiotic resistance and loss of antibiotic binding. The data also suggest that GE82832/dityromycin inhibits EF-G-catalyzed translocation by disrupting a critical contact between EF-G and S12 that is required to stabilize the posttranslocational conformation of EF-G, thereby preventing the ribosome-EF-G complex from entering a conformation productive for translocation.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Fator 2 de Elongação de Peptídeos/metabolismo , Peptídeos/farmacologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Sítios de Ligação , Escherichia coli/efeitos dos fármacos , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Fator 2 de Elongação de Peptídeos/química , Fator 2 de Elongação de Peptídeos/genética , Ligação Proteica , Thermus thermophilus/efeitos dos fármacosRESUMO
Upon high throughput screening of 6700 microbial fermentation extracts, we discovered a compound, designated orthoformimycin, capable of inhibiting protein synthesis in vitro with high efficiency. The molecule, whose structure was elucidated by chemical, spectrometric, and spectroscopic methods, contains an unusual orthoformate moiety (hence the name) and belongs to a novel class of translation inhibitors. This antibiotic does not affect any function of the 30S ribosomal subunit but binds to the 50S subunit causing inhibition of translation elongation and yielding polypeptide products of reduced length. Analysis by fluorescence stopped flow kinetics revealed that EF-G-dependent mRNA translocation is inhibited by orthoformimycin, whereas, surprisingly, translocation of the aminoacyl-tRNA seems to be unaffected.
Assuntos
Antibacterianos/química , Descoberta de Drogas , Formiatos/química , Fungos/química , Biossíntese de Proteínas/efeitos dos fármacos , Streptomyces/efeitos dos fármacos , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Formiatos/isolamento & purificação , Formiatos/farmacologia , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Fator G para Elongação de Peptídeos/metabolismo , Streptomyces/metabolismoRESUMO
Furvina®, also denominated G1 (MW 297), is a synthetic nitrovinylfuran [2-bromo-5-(2-bromo-2-nitrovinyl)-furan] antibiotic with a broad antimicrobial spectrum. An ointment (Dermofural®) containing G1 as the only active principle is currently marketed in Cuba and successfully used to treat dermatological infections. Here we describe the molecular target and mechanism of action of G1 in bacteria and demonstrate that in vivo G1 preferentially inhibits protein synthesis over RNA, DNA and cell wall synthesis. Furthermore, we demonstrate that G1 targets the small ribosomal subunit, binds at or near the P-decoding site and inhibits its function interfering with the ribosomal binding of fMet-tRNA during 30S initiation complex (IC) formation ultimately inhibiting translation. Notably, this G1 inhibition displays a bias for the nature (purine vs. pyrimidine) of the 3'-base of the codon, occurring efficiently only when the mRNA directing 30S IC formation and translation contains the canonical AUG initiation triplet or the rarely found AUA triplet, but hardly occurs when the mRNA start codon is either one of the non-canonical triplets AUU or AUC. This codon discrimination by G1 is reminiscent, though of opposite type of that displayed by IF3 in its fidelity function, and remarkably does not occur in the absence of this factor.
Assuntos
Antibacterianos/farmacologia , Códon de Iniciação , Furanos/farmacologia , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Subunidades Ribossômicas Menores de Bactérias/efeitos dos fármacos , Compostos de Vinila/farmacologia , Sítios de Ligação , Subunidades Ribossômicas Menores de Bactérias/químicaRESUMO
The structure of GE82832, a translocation inhibitor produced by a soil microorganism, is shown to be highly related to that of dityromycin, a bicyclodecadepsipeptide antibiotic discovered long ago whose characterization had never been pursued beyond its structural elucidation. GE82832 and dityromycin were shown to interfere with both aminoacyl-tRNA and mRNA movement and with the Pi release occurring after ribosome- and EF-G-dependent GTP hydrolysis. These findings and the unusual ribosomal localization of GE82832/dityromycin near protein S13 suggest that the mechanism of inhibition entails an interference with the rotation of the 30S subunit "head" which accompanies the ribosome-unlocking step of translocation.
Assuntos
Antibacterianos/química , Antibacterianos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Actinomycetales/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/farmacologia , Translocação Bacteriana/efeitos dos fármacos , Guanosina Trifosfato/metabolismo , Cinética , Modelos Moleculares , Estrutura Molecular , Fator G para Elongação de Peptídeos/metabolismo , Peptídeos/farmacologia , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Microbiologia do Solo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Translation initiation factor IF2 is a guanine nucleotide-binding protein. The free energy change associated with guanosine triphosphate hydrolase (GTPase) activity of these proteins is believed to be the driving force allowing them to perform their functions as molecular switches. We examined role and relevance of IF2 GTPase and demonstrate that an Escherichia coli IF2 mutant bearing a single amino acid substitution (E571K) in its 30S binding domain (IF2-G3) can perform in vitro all individual translation initiation functions of wild type (wt) IF2 and supports faithful messenger RNA translation, despite having a reduced affinity for the 30S subunit and being completely inactive in GTP hydrolysis. Furthermore, the corresponding GTPase-null mutant of Bacillus stearothermophilus (E424K) can replace in vivo wt IF2 allowing an E. coli infB null mutant to grow with almost wt duplication times. Following the E571K (and E424K) mutation, which likely disrupts hydrogen bonding between subdomains G2 and G3, IF2 acquires a guanosine diphosphate (GDP)-like conformation, no longer responsive to GTP binding thereby highlighting the importance of interdomain communication in IF2. Our data underlie the importance of GTP as an IF2 ligand in the early initiation steps and the dispensability of the free energy generated by the IF2 GTPase in the late events of the translation initiation pathway.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Iniciação Traducional da Cadeia Peptídica , Fator de Iniciação 2 em Procariotos/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , GTP Fosfo-Hidrolases/genética , Geobacillus stearothermophilus/genética , Hidrólise , Mutação , Fator de Iniciação 2 em Procariotos/química , Fator de Iniciação 2 em Procariotos/genética , Estrutura Terciária de Proteína , Subunidades Ribossômicas Menores de Bactérias/metabolismoRESUMO
Since their introduction in therapy, antibiotics have played an essential role in human society, saving millions of lives, allowing safe surgery, organ transplants, cancer therapy. Antibiotics have also helped to elucidate several biological mechanisms and boosted the birth and growth of pharmaceutical companies, generating profits and royalties. The golden era of antibiotics and the scientific and economical drive of big pharma towards these molecules is long gone, but the need for effective antibiotics is increased as their pipelines dwindle and multi-resistant pathogenic strains spread. Here we outline some strategies that could help meet this emergency and list promising new targets.
Assuntos
Antibacterianos , Descoberta de Drogas/métodos , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Humanos , Biossíntese de Proteínas/efeitos dos fármacosRESUMO
While bacterial protein synthesis is the target of about half of the known antibiotics, the great structural-functional complexity of the translational machinery still offers remarkable opportunities for identifying novel and specific inhibitors of unexploited targets. We designed a knowledge-based in vitro translation assay to identify inhibitors selectively targeting the bacterial or the yeast translational apparatus, preferentially blocking the early steps of protein synthesis. Using a natural-like, "universal" model mRNA and cell-free extracts prepared from Eschericha coli, Saccharomyces cerevisiae, and HeLa cells, we were able to translate, with comparable yields in the three systems, the immunogenic peptide encoded by this "universal" mRNA. The immuno-enzymatic quantification of the translated peptide in the presence of a potential inhibitor can identify a selective bacterial or fungal inhibitor inactive in the human system. When applied to the high-throughput screening (HTS) of a library of approximately 25,000 natural products, this assay led to the identification of two novel and specific inhibitors of bacterial translation.
Assuntos
Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/isolamento & purificação , Sequência de Aminoácidos , Sistema Livre de Células , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Inibidores da Síntese de Proteínas/análise , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcrição GênicaRESUMO
BACKGROUND: Translation initiation is a basic and universal biological process that employs significantly different components and displays substantially different mechanisms in bacterial, archaeal and eukaryotic cells. A large amount of detailed mechanistic and structural information on the bacterial translation initiation apparatus has been uncovered in recent years. OBJECTIVE: to understand which translation initiation steps could represent a novel or underexploited target for the discovery of new and specific antibacterial drugs. METHODS: Brief descriptions of the properties and mechanism of action of the major antibiotics that have a documented direct inhibitory effect on bacterial translation initiation are presented. RESULTS/CONCLUSIONS: Considerations and predictions concerning a future scenario for research and identification of bacterial translation initiation inhibitors are presented.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Fenômenos Fisiológicos Bacterianos/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Iniciação Traducional da Cadeia Peptídica/fisiologia , Fatores de Iniciação de Peptídeos/efeitos dos fármacos , Fatores de Iniciação de Peptídeos/metabolismoRESUMO
This chapter presents methods and protocols suitable for the identification and characterization of inhibitors of the prokaryotic and/or eukaryotic translational apparatus as a whole or targeting specific, underexploited targets of the bacterial protein synthetic machinery such as translation initiation and aminoacylation. Some of the methods described have been used successfully for the high-throughput screening of libraries of natural or synthetic compounds and make use of model "universal" mRNAs that can be translated with similar efficiency by cellfree extracts of bacterial, yeast, and HeLa cells. Other methods presented here are suitable for secondary screening tests aimed at identifying a specific target of an antibiotic within the translational pathway of prokaryotic cells.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores da Síntese de Ácido Nucleico/isolamento & purificação , Biossíntese de Proteínas/efeitos dos fármacos , Animais , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/metabolismo , Sistema Livre de Células/metabolismo , Células Cultivadas , Técnicas de Laboratório Clínico , Humanos , Luciferases de Renilla/genética , Luciferases de Renilla/metabolismo , Inibidores da Síntese de Ácido Nucleico/análise , Fator de Iniciação 2 em Procariotos/antagonistas & inibidores , Fator de Iniciação 2 em Procariotos/fisiologia , Proteínas de Ligação ao Cap de RNA/fisiologia , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Aminoacilação de RNA de Transferência/efeitos dos fármacos , Leveduras/genética , Leveduras/metabolismoRESUMO
There is a need to develop novel antibiotics for treating infections caused by multiresistant pathogens. Notwithstanding a plethora of novel targets and intensive high-throughput screening, conventional chemistry has yet to deliver these badly needed new drugs. Microorganisms have provided many of the existing antibiotics, but there is a general feeling that the large majority of compounds have already been discovered. Novel assays, used to screen common microbes, can provide novel structural scaffolds for antibiotic discovery. However, the highest impact may come from unexplored microbial sources. Fortunately, there is plenty of previously undescribed, antibiotic-producing bacteria in the environment.
RESUMO
GE82832, a secondary metabolite produced by Streptosporangium cinnabarinum (strain GE82832), has been identified as a translational inhibitor by in vitro screening of a library of natural products. Secondary functional tests specific for individual steps of the translational pathway demonstrated that translocation is the specific target of GE82832. Chemical probing in situ demonstrated that this antibiotic protects bases A1324 and A1333 and exposes C1336 of 16S rRNA, thereby indicating that its binding site is located on the head of the 30S ribosomal subunit. The ribosomal location of GE82832, near ribosomal protein S13 and G1338, two elements of the small subunit that are part of or close to the B1a intrasubunit bridge, suggests that translocation inhibition results from an altered dynamics of 30S-50S ribosomal subunit interaction.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Transporte Proteico/efeitos dos fármacos , Actinomycetales/química , Proteínas de Bactérias/genética , Modelos Moleculares , Biossíntese de Proteínas/efeitos dos fármacos , Conformação Proteica , Puromicina/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Ribossomos/química , Ribossomos/metabolismoRESUMO
In the course of a microbial product screening aimed at the discovery of novel antibiotics acting on bacterial protein synthesis, a complex of three structurally related tetrapeptides, namely, GE81112 factors A, B, and B1, was isolated from a Streptomyces sp. The screening was based on a cell-free assay of bacterial protein synthesis driven by a model mRNA containing natural initiation signals. In this study we report the production, isolation, and structure determination of these novel, potent and selective inhibitors of cell-free bacterial protein synthesis, which stably bind the 30S ribosomal subunit and inhibit the formation of fMet-puromycin. They did not inhibit translation by yeast ribosomes in vitro. Spectroscopic analyses revealed that they are tetrapeptides constituted by uncommon amino acids. While GE81112 factors A, B, and B1 were effective in inhibiting bacterial protein synthesis in vitro, they were less active against Gram-positive and Gram-negative bacterial cells. Cells grown in minimal medium were more susceptible to the compounds than those grown in rich medium, and this is most likely due to competition or regulation by medium components during peptide uptake. The novelty of the chemical structure and of the specific mode of action on the initiation phase of bacterial protein synthesis makes GE81112 a unique scaffold for designing new drugs.