RESUMEN
Growing resistance toward ribosome-targeting macrolide antibiotics has limited their clinical utility and urged the search for superior compounds. Macrolones are synthetic macrolide derivatives with a quinolone side chain, structurally similar to DNA topoisomerase-targeting fluoroquinolones. While macrolones show enhanced activity, their modes of action have remained unknown. Here, we present the first structures of ribosome-bound macrolones, showing that the macrolide part occupies the macrolide-binding site in the ribosomal exit tunnel, whereas the quinolone moiety establishes new interactions with the tunnel. Macrolones efficiently inhibit both the ribosome and DNA topoisomerase in vitro. However, in the cell, they target either the ribosome or DNA gyrase or concurrently both of them. In contrast to macrolide or fluoroquinolone antibiotics alone, dual-targeting macrolones are less prone to select resistant bacteria carrying target-site mutations or to activate inducible macrolide resistance genes. Furthermore, because some macrolones engage Erm-modified ribosomes, they retain activity even against strains with constitutive erm resistance genes.
RESUMEN
The Proline-rich Antimicrobial Peptide (PrAMP) apidaecin (Api) inhibits translation by binding in the ribosomal nascent peptide exit tunnel, trapping release factors RF1 or RF2, and arresting ribosomes at stop codons. To explore the extent of sequence variations of the native 18-amino acid Api that allows it to preserve its activity, we screened a library of synthetic mutant Api genes expressed in bacterial cells, resulting in nearly 350000 peptide variants with multiple substitutions. By applying orthogonal negative and positive selection strategies, we identified a number of multi-substituted Api variants capable of arresting ribosomes at stop codons. Our findings underscore the critical contribution of specific amino acid residues of the peptide for its on-target function while significantly expanding the variety of PrAMPs acting on the terminating ribosome. Additionally, some of the tested synthesized multi-substituted Api variants exhibit improved antibacterial activity compared to that of the wild type PrAMP and may constitute the starting point to develop clinically useful antimicrobials.
RESUMEN
The paenilamicins are a group of hybrid non-ribosomal peptide-polyketide compounds produced by the honey bee pathogen Paenibacillus larvae that display activity against Gram-positive pathogens, such as Staphylococcus aureus. While paenilamicins have been shown to inhibit protein synthesis, their mechanism of action has remained unclear. Here, we have determined structures of the paenilamicin PamB2 stalled ribosomes, revealing a unique binding site on the small 30S subunit located between the A- and P-site tRNAs. In addition to providing a precise description of interactions of PamB2 with the ribosome, the structures also rationalize the resistance mechanisms utilized by P. larvae. We could further demonstrate that PamB2 interferes with the translocation of mRNA and tRNAs through the ribosome during translation elongation, and that this inhibitory activity is influenced by the presence of modifications at position 37 of the A-site tRNA. Collectively, our study defines the paenilamicins as a new class of context-specific translocation inhibitors.
RESUMEN
Expression of the Escherichia coli tnaCAB operon, responsible for L-tryptophan (L-Trp) transport and catabolism, is regulated by L-Trp-directed translation arrest and the ribosome arresting peptide TnaC. The function of TnaC relies on conserved residues distributed throughout the peptide, which are involved in forming an L-Trp binding site at the ribosome exit tunnel and inhibiting the ribosome function. We aimed to understand whether nonconserved amino acids surrounding these critical conserved residues play a functional role in TnaC-mediated ribosome arrest. We have isolated two intragenic suppressor mutations that restore arrest function of TnaC mutants; one of these mutations is located near the L-Trp binding site, while the other mutation is located near the ribosome active site. We used reporter gene fusions to show that both suppressor mutations have similar effects on TnaC mutants at the conserved residues involved in forming a free L-Trp binding site. However, they diverge in suppressing loss-of-function mutations in a conserved TnaC residue at the ribosome active site. With ribosome toeprinting assays, we determined that both suppressor mutations generate TnaC peptides, which are highly sensitive to L-Trp. Puromycin-challenge assays with isolated arrested ribosomes indicate that both TnaC suppressor mutants are resistant to peptidyl-tRNA cleavage by puromycin in the presence of L-Trp; however, they differ in their resistance to puromycin in the absence of L-Trp. We propose that the TnaC peptide two functionally distinct segments, a sensor domain and a stalling domain, and that the functional versatility of these domains is fine-tuned by the nature of their surrounding nonconserved residues.
Asunto(s)
Escherichia coli , Biosíntesis de Proteínas , Ribosomas , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Péptidos/metabolismo , Puromicina , Ribosomas/metabolismoRESUMEN
Rescuing stalled ribosomes often involves their splitting into subunits. In many bacteria, the resultant large subunits bearing peptidyl-tRNAs are processed by the ribosome-associated quality control (RQC) apparatus that extends the C termini of the incomplete nascent polypeptides with polyalanine tails to facilitate their degradation. Although the tailing mechanism is well established, it is unclear how the nascent polypeptides are cleaved off the tRNAs. We show that peptidyl-tRNA hydrolase (Pth), the known role of which has been to hydrolyze ribosome-free peptidyl-tRNA, acts in concert with RQC factors to release nascent polypeptides from large ribosomal subunits. Dislodging from the ribosomal catalytic center is required for peptidyl-tRNA hydrolysis by Pth. Nascent protein folding may prevent peptidyl-tRNA retraction and interfere with the peptide release. However, oligoalanine tailing makes the peptidyl-tRNA ester bond accessible for Pth-catalyzed hydrolysis. Therefore, the oligoalanine tail serves not only as a degron but also as a facilitator of Pth-catalyzed peptidyl-tRNA hydrolysis.
Asunto(s)
Hidrolasas de Éster Carboxílico , Péptidos , Ribosomas , Ribosomas/metabolismo , Péptidos/genética , Bacterias/genética , Control de Calidad , Biosíntesis de ProteínasRESUMEN
The proline-rich antimicrobial peptide (PrAMP) Drosocin (Dro) from fruit flies shows sequence similarity to other PrAMPs that bind to the ribosome and inhibit protein synthesis by varying mechanisms. The target and mechanism of action of Dro, however, remain unknown. Here we show that Dro arrests ribosomes at stop codons, probably sequestering class 1 release factors associated with the ribosome. This mode of action is comparable to that of apidaecin (Api) from honeybees, making Dro the second member of the type II PrAMP class. Nonetheless, analysis of a comprehensive library of endogenously expressed Dro mutants shows that the interactions of Dro and Api with the target are markedly distinct. While only a few C-terminal amino acids of Api are critical for binding, the interaction of Dro with the ribosome relies on multiple amino acid residues distributed throughout the PrAMP. Single-residue substitutions can substantially enhance the on-target activity of Dro.
Asunto(s)
Péptidos Antimicrobianos , Biosíntesis de Proteínas , Animales , Escherichia coli/metabolismo , Glicopéptidos/química , Drosophila/química , Drosophila/metabolismoRESUMEN
Orthosomycin antibiotics inhibit protein synthesis by binding to the large ribosomal subunit in the tRNA accommodation corridor, which is traversed by incoming aminoacyl-tRNAs. Structural and biochemical studies suggested that orthosomycins block accommodation of any aminoacyl-tRNAs in the ribosomal A-site. However, the mode of action of orthosomycins in vivo remained unknown. Here, by carrying out genome-wide analysis of antibiotic action in bacterial cells, we discovered that orthosomycins primarily inhibit the ribosomes engaged in translation of specific amino acid sequences. Our results reveal that the predominant sites of orthosomycin-induced translation arrest are defined by the nature of the incoming aminoacyl-tRNA and likely by the identity of the two C-terminal amino acid residues of the nascent protein. We show that nature exploits this antibiotic-sensing mechanism for directing programmed ribosome stalling within the regulatory open reading frame, which may control expression of an orthosomycin-resistance gene in a variety of bacterial species.
Asunto(s)
Antibacterianos , Ribosomas , Antibacterianos/farmacología , Antibacterianos/metabolismo , Ribosomas/metabolismo , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Secuencia de Aminoácidos , Biosíntesis de ProteínasRESUMEN
Streptococcus pneumoniae, an opportunistic human pathogen, is the leading cause of community-acquired pneumonia and an agent of otitis media, septicemia, and meningitis. Although genomic and transcriptomic studies of S. pneumoniae have provided detailed perspectives on gene content and expression programs, they have lacked information pertaining to the translational landscape, particularly at a resolution that identifies commonly overlooked small open reading frames (sORFs), whose importance is increasingly realized in metabolism, regulation, and virulence. To identify protein-coding sORFs in S. pneumoniae, antibiotic-enhanced ribosome profiling was conducted. Using translation inhibitors, 114 novel sORFs were detected, and the expression of a subset of them was experimentally validated. Two loci associated with virulence and quorum sensing were examined in deeper detail. One such sORF, rio3, overlaps with the noncoding RNA srf-02 that was previously implicated in pathogenesis. Targeted mutagenesis parsing rio3 from srf-02 revealed that rio3 is responsible for the fitness defect seen in a murine nasopharyngeal colonization model. Additionally, two novel sORFs located adjacent to the quorum sensing receptor rgg1518 were found to impact regulatory activity. Our findings emphasize the importance of sORFs present in the genomes of pathogenic bacteria and underscore the utility of ribosome profiling for identifying the bacterial translatome. IMPORTANCE This work employed pleuromutilin-assisted ribosome profiling using retapamulin (Ribo-RET) to identify genome-wide translation start sites in the human pathogen Streptococcus pneumoniae. We identified 114 unannotated intergenic small open reading frames (sORFs). The described procedures and data sets provide a model for microbiologists seeking to explore the translational landscape of bacteria. The biological roles of four sORF examples are characterized: two control the regulation of a cell-cell communication (quorum sensing) system, one contributes to the ability of S. pneumoniae to colonize the upper respiratory tract of mice, and a fourth governs the translation of PrfB, a protein enabling ribosome release at stop codons. We propose that Ribo-RET is a valuable approach to identifying unstudied microproteins and difficult-to-find pheromone genes used by Gram-positive organisms, whose genomes are replete with pheromone receptors.
Asunto(s)
Percepción de Quorum , Streptococcus pneumoniae , Animales , Humanos , Ratones , Sistemas de Lectura Abierta , Percepción de Quorum/genética , Ribosomas/genética , Ribosomas/metabolismo , Streptococcus pneumoniae/genética , VirulenciaRESUMEN
The antibiotic linezolid, the first clinically approved member of the oxazolidinone class, inhibits translation of bacterial ribosomes by binding to the peptidyl transferase center. Recent work has demonstrated that linezolid does not inhibit peptide bond formation at all sequences but rather acts in a context-specific manner, namely when alanine occupies the penultimate position of the nascent chain. However, the molecular basis for context-specificity has not been elucidated. Here we show that the second-generation oxazolidinone radezolid also induces stalling with a penultimate alanine, and we determine high-resolution cryo-EM structures of linezolid- and radezolid-stalled ribosome complexes to explain their mechanism of action. These structures reveal that the alanine side chain fits within a small hydrophobic crevice created by oxazolidinone, resulting in improved ribosome binding. Modification of the ribosome by the antibiotic resistance enzyme Cfr disrupts stalling due to repositioning of the modified nucleotide. Together, our findings provide molecular understanding for the context-specificity of oxazolidinones.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Oxazolidinonas/química , Oxazolidinonas/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Alanina/química , Sitios de Unión , Microscopía por Crioelectrón , Linezolid/química , Linezolid/farmacología , Modelos Moleculares , Peptidil Transferasas/metabolismo , ARN Ribosómico/química , ARN Ribosómico/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Ribosomas/ultraestructuraRESUMEN
Ribosome-targeting antibiotics serve as powerful antimicrobials and as tools for studying the ribosome, the catalytic peptidyl transferase center (PTC) of which is targeted by many drugs. The classic PTC-acting antibiotic chloramphenicol (CHL) and the newest clinically significant linezolid (LZD) were considered indiscriminate inhibitors of protein synthesis that cause ribosome stalling at every codon of every gene being translated. However, recent discoveries have shown that CHL and LZD preferentially arrest translation when the ribosome needs to polymerize particular amino acid sequences. The molecular mechanisms that underlie the context-specific action of ribosome inhibitors are unknown. Here we present high-resolution structures of ribosomal complexes, with or without CHL, carrying specific nascent peptides that support or negate the drug action. Our data suggest that the penultimate residue of the nascent peptide directly modulates antibiotic affinity to the ribosome by either establishing specific interactions with the drug or by obstructing its proper placement in the binding site.
Asunto(s)
Cloranfenicol/química , Cloranfenicol/farmacología , Peptidil Transferasas/antagonistas & inhibidores , Antibacterianos/química , Antibacterianos/farmacología , Sitios de Unión , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Cinética , Modelos Moleculares , Conformación Proteica , Inhibidores de la Síntesis de la Proteína/química , Inhibidores de la Síntesis de la Proteína/farmacología , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/metabolismo , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Electricidad Estática , Thermus thermophilus/efectos de los fármacos , Thermus thermophilus/metabolismoRESUMEN
Kasugamycin (KSG) is an aminoglycoside antibiotic widely used in agriculture and exhibits considerable medical potential. Previous studies suggested that KSG interferes with translation by blocking binding of canonical messenger RNA (mRNA) and initiator transfer tRNA (tRNA) to the small ribosomal subunit, thereby preventing initiation of protein synthesis. Here, by using genome-wide approaches, we show that KSG can interfere with translation even after the formation of the 70S initiation complex on mRNA, as the extent of KSG-mediated translation inhibition correlates with increased occupancy of start codons by 70S ribosomes. Even at saturating concentrations, KSG does not completely abolish translation, allowing for continuing expression of some Escherichia coli proteins. Differential action of KSG significantly depends on the nature of the mRNA residue immediately preceding the start codon, with guanine in this position being the most conducive to inhibition by the drug. In addition, the activity of KSG is attenuated by translational coupling as genes whose start codons overlap with the coding regions or the stop codons of the upstream cistrons tend to be less susceptible to drug-mediated inhibition. Altogether, our findings reveal KSG as an example of a small ribosomal subunit-targeting antibiotic with a well-pronounced context specificity of action.
Asunto(s)
Aminoglicósidos/farmacología , Sitios de Unión , Iniciación de la Cadena Peptídica Traduccional/efectos de los fármacos , ARN Mensajero/genética , Ribosomas/metabolismo , Aminoglicósidos/química , Codón Iniciador , Estructura Molecular , Sistemas de Lectura Abierta , Unión Proteica , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/química , ARN Mensajero/metabolismo , Ribosomas/química , Relación Estructura-ActividadRESUMEN
Small proteins encoded by open reading frames (ORFs) shorter than 50 codons (small ORFs [sORFs]) are often overlooked by annotation engines and are difficult to characterize using traditional biochemical techniques. Ribosome profiling has tremendous potential to empirically improve the annotations of prokaryotic genomes. Recent improvements in ribosome profiling methods for bacterial model organisms have revealed many new sORFs in well-characterized genomes. Antibiotics that trap ribosomes just after initiation have played a key role in these developments by allowing the unambiguous identification of the start codons (and, hence, the reading frame) for novel ORFs. Here, we describe these new methods and highlight critical controls and considerations for adapting ribosome profiling to different prokaryotic species.
Asunto(s)
Antibacterianos/farmacología , Bacterias/metabolismo , Sistemas de Lectura Abierta , Ribosomas , Bacterias/genética , Codón , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/fisiología , Iniciación de la Cadena Peptídica Traduccional , Terminación de la Cadena Péptídica Traduccional , ARN Bacteriano , ARN RibosómicoRESUMEN
Free L-tryptophan (L-Trp) stalls ribosomes engaged in the synthesis of TnaC, a leader peptide controlling the expression of the Escherichia coli tryptophanase operon. Despite extensive characterization, the molecular mechanism underlying the recognition and response to L-Trp by the TnaC-ribosome complex remains unknown. Here, we use a combined biochemical and structural approach to characterize a TnaC variant (R23F) with greatly enhanced sensitivity for L-Trp. We show that the TnaC-ribosome complex captures a single L-Trp molecule to undergo termination arrest and that nascent TnaC prevents the catalytic GGQ loop of release factor 2 from adopting an active conformation at the peptidyl transferase center. Importantly, the L-Trp binding site is not altered by the R23F mutation, suggesting that the relative rates of L-Trp binding and peptidyl-tRNA cleavage determine the tryptophan sensitivity of each variant. Thus, our study reveals a strategy whereby a nascent peptide assists the ribosome in detecting a small metabolite.
Asunto(s)
Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Iniciación de la Cadena Peptídica Traduccional , Ribosomas/genética , Triptófano/química , Sustitución de Aminoácidos , Sitios de Unión , Microscopía por Crioelectrón , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Mutación , Operón , Terminación de la Cadena Péptídica Traduccional , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Ribosomas/ultraestructura , Triptófano/metabolismoRESUMEN
Macrolides and ketolides comprise a family of clinically important antibiotics that inhibit protein synthesis by binding within the exit tunnel of the bacterial ribosome. While these antibiotics are known to interrupt translation at specific sequence motifs, with ketolides predominantly stalling at Arg/Lys-X-Arg/Lys motifs and macrolides displaying a broader specificity, a structural basis for their context-specific action has been lacking. Here, we present structures of ribosomes arrested during the synthesis of an Arg-Leu-Arg sequence by the macrolide erythromycin (ERY) and the ketolide telithromycin (TEL). Together with deep mutagenesis and molecular dynamics simulations, the structures reveal how ERY and TEL interplay with the Arg-Leu-Arg motif to induce translational arrest and illuminate the basis for the less stringent sequence-specific action of ERY over TEL. Because programmed stalling at the Arg/Lys-X-Arg/Lys motifs is used to activate expression of antibiotic resistance genes, our study also provides important insights for future development of improved macrolide antibiotics.
Asunto(s)
Antibacterianos/farmacología , Cetólidos/farmacología , Macrólidos/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Antibacterianos/química , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/enzimología , Bacillus subtilis/genética , Sitios de Unión/genética , Microscopía por Crioelectrón , Farmacorresistencia Microbiana/genética , Eritromicina/química , Eritromicina/farmacología , Genes Bacterianos , Cetólidos/química , Cetólidos/farmacocinética , Macrólidos/química , Metiltransferasas/química , Metiltransferasas/genética , Metiltransferasas/metabolismo , Simulación de Dinámica Molecular , Mutagénesis Insercional , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/química , Ribosomas/efectos de los fármacosRESUMEN
Macrolide antibiotics bind in the nascent peptide exit tunnel of the bacterial ribosome and prevent polymerization of specific amino acid sequences, selectively inhibiting translation of a subset of proteins. Because preventing translation of individual proteins could be beneficial for the treatment of human diseases, we asked whether macrolides, if bound to the eukaryotic ribosome, would retain their context- and protein-specific action. By introducing a single mutation in rRNA, we rendered yeast Saccharomyces cerevisiae cells sensitive to macrolides. Cryo-EM structural analysis showed that the macrolide telithromycin binds in the tunnel of the engineered eukaryotic ribosome. Genome-wide analysis of cellular translation and biochemical studies demonstrated that the drug inhibits eukaryotic translation by preferentially stalling ribosomes at distinct sequence motifs. Context-specific action markedly depends on the macrolide structure. Eliminating macrolide-arrest motifs from a protein renders its translation macrolide-tolerant. Our data illuminate the prospects of adapting macrolides for protein-selective translation inhibition in eukaryotic cells.
Asunto(s)
Antibacterianos/farmacología , Macrólidos/farmacología , Ribosomas/efectos de los fármacos , Antibacterianos/química , Sitios de Unión , Microscopía por Crioelectrón , Células Eucariotas/efectos de los fármacos , Células Eucariotas/metabolismo , Humanos , Macrólidos/química , Modelos Moleculares , Mutación , Unión Proteica , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/química , Inhibidores de la Síntesis de la Proteína/farmacología , ARN de Hongos/genética , ARN Ribosómico/genética , Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biosíntesis , Relación Estructura-ActividadRESUMEN
The knowledge of translation start sites is crucial for annotation of genes in bacterial genomes. However, systematic mapping of start codons in bacterial genes has mainly relied on predictions based on protein conservation and mRNA sequence features which, although useful, are not always accurate. We recently found that the pleuromutilin antibiotic retapamulin (RET) is a specific inhibitor of translation initiation that traps ribosomes specifically at start codons, and we used it in combination with ribosome profiling to map start codons in the Escherichia coli genome. This genome-wide strategy, that was named Ribo-RET, not only verifies the position of start codons in already annotated genes but also enables identification of previously unannotated open reading frames and reveals the presence of internal start sites within genes. Here, we provide a detailed Ribo-RET protocol for E. coli. Ribo-RET can be adapted for mapping the start codons of the protein-coding sequences in a variety of bacterial species.
Asunto(s)
Codón Iniciador , Biología Computacional/métodos , Escherichia coli/genética , Ribosomas/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Codón Iniciador/efectos de los fármacos , Diterpenos/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Genoma Bacteriano , Anotación de Secuencia Molecular , Sistemas de Lectura Abierta , Biosíntesis de Proteínas/efectos de los fármacosRESUMEN
Apidaecin (Api), an unmodified 18-amino-acid-long proline-rich antibacterial peptide produced by bees, has been recently described as a specific inhibitor of translation termination. It invades the nascent peptide exit tunnel of the postrelease ribosome and traps the release factors preventing their recycling. Api binds in the exit tunnel in an extended conformation that matches the placement of a nascent polypeptide and establishes multiple contacts with ribosomal RNA (rRNA) and ribosomal proteins. Which of these interactions are critical for Api's activity is unknown. We addressed this problem by analyzing the activity of all possible single-amino-acid substitutions of the Api variants synthesized in the bacterial cell. By conditionally expressing the engineered api gene, we generated Api directly in the bacterial cytosol, thereby bypassing the need for importing the peptide from the medium. The endogenously expressed Api, as well as its N-terminally truncated mutants, retained the antibacterial properties and the mechanism of action of the native peptide. Taking advantage of the Api expression system and next-generation sequencing, we mapped in one experiment all the single-amino-acid substitutions that preserve or alleviate the on-target activity of the Api mutants. Analysis of the inactivating mutations made it possible to define the pharmacophore of Api involved in critical interactions with the ribosome, transfer RNA (tRNA), and release factors. We also identified the Api segment that tolerates a variety of amino acid substitutions; alterations in this segment could be used to improve the pharmacological properties of the antibacterial peptide.
Asunto(s)
Péptidos Catiónicos Antimicrobianos , Escherichia coli , Terminación de la Cadena Péptídica Traduccional/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína , Sustitución de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/farmacología , Abejas , Escherichia coli/genética , Escherichia coli/metabolismo , Mutación Missense , Inhibidores de la Síntesis de la Proteína/química , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Ribosómico/química , ARN Ribosómico/genética , ARN Ribosómico/metabolismoRESUMEN
Biochemical studies suggested that the antimicrobial peptide apidaecin (Api) inhibits protein synthesis by binding in the nascent peptide exit tunnel and trapping the release factor associated with a terminating ribosome. The mode of Api action in bacterial cells had remained unknown. Here genome-wide analysis reveals that in bacteria, Api arrests translating ribosomes at stop codons and causes pronounced queuing of the trailing ribosomes. By sequestering the available release factors, Api promotes pervasive stop codon bypass, leading to the expression of proteins with C-terminal extensions. Api-mediated translation arrest leads to the futile activation of the ribosome rescue systems. Understanding the unique mechanism of Api action in living cells may facilitate the development of new medicines and research tools for genome exploration.