RESUMEN
In the absence of elongation factor EF-G, ribosomes undergo spontaneous, thermally driven fluctuation between the pre-translocation (classical) and intermediate (hybrid) states of translocation. These fluctuations do not result in productive mRNA translocation. Extending previous findings that the antibiotic sparsomycin induces translocation, we identify additional peptidyl transferase inhibitors that trigger productive mRNA translocation. We find that antibiotics that bind the peptidyl transferase A site induce mRNA translocation, whereas those that do not occupy the A site fail to induce translocation. Using single-molecule FRET, we show that translocation-inducing antibiotics do not accelerate intersubunit rotation, but act solely by converting the intrinsic, thermally driven dynamics of the ribosome into translocation. Our results support the idea that the ribosome is a Brownian ratchet machine, whose intrinsic dynamics can be rectified into unidirectional translocation by ligand binding.
Asunto(s)
Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Transporte de ARN/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , Subunidades Ribosómicas Grandes Bacterianas/efectos de los fármacos , Antibacterianos/metabolismo , Cloranfenicol/metabolismo , Cloranfenicol/farmacología , Clindamicina/metabolismo , Clindamicina/farmacología , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Lincomicina/metabolismo , Lincomicina/farmacología , Factor G de Elongación Peptídica/efectos de los fármacos , Factor G de Elongación Peptídica/metabolismo , Peptidil Transferasas/efectos de los fármacos , Peptidil Transferasas/metabolismo , ARN Bacteriano/efectos de los fármacos , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , ARN de Transferencia/efectos de los fármacos , ARN de Transferencia/metabolismo , Subunidades Ribosómicas Grandes Bacterianas/metabolismo , Esparsomicina/metabolismo , Esparsomicina/farmacologíaRESUMEN
Sparsomycin is an antibiotic that targets the peptidyl transferase center of the ribosome and has the ability to promote ribosomal translocation in the absence of EF-G and GTP. Here we show that changes in the configurations at the two chiral centers of sparsomycin, especially at the chiral carbon, can greatly affect its capability to promote ribosomal translocation. More importantly, the incorporation of the pseudo-uracil moiety of sparsomycin into linezolid through a covalent linkage conferred on linezolid derivatives the ability to promote translocation, thus indicating the importance of interactions between this pseudo-uracil moiety, rRNA, and tRNA for promoting translocation. In addition, these translocation promoters can also effectively inhibit spontaneous reverse translocation; this suggests that they might promote forward translocation by trapping the ribosome in the post-translocation state and shifting the equilibrium between the pre- and post-translocation ribosome in the forward direction.
Asunto(s)
Acetamidas/farmacología , Antibacterianos/farmacología , Oxazolidinonas/farmacología , Ribosomas/metabolismo , Esparsomicina/farmacología , Acetamidas/química , Antibacterianos/química , Transporte Biológico , Linezolid , Modelos Moleculares , Oxazolidinonas/química , Esparsomicina/químicaRESUMEN
From the X-ray crystal structures of linezolid and the non-selective antibiotic sparsomycin, we have derived a new family of hybrid oxazolidinones. From this initial compound set we have developed a new biaryloxazolidinone scaffold that shows both potent antimicrobial activity as well as selective inhibition of ribosomal translation. The synthesis of these compounds is outlined.
Asunto(s)
Antibacterianos/síntesis química , Antibacterianos/farmacología , Oxazolidinonas/síntesis química , Oxazolidinonas/farmacología , Acetamidas/farmacología , Administración Oral , Antibacterianos/química , Cristalografía por Rayos X , Diseño de Fármacos , Escherichia coli/efectos de los fármacos , Haemophilus influenzae/efectos de los fármacos , Linezolid , Pruebas de Sensibilidad Microbiana , Conformación Molecular , Estructura Molecular , Moraxella catarrhalis/efectos de los fármacos , Oxazolidinonas/química , Biosíntesis de Proteínas/efectos de los fármacos , Esparsomicina/farmacología , Relación Estructura-ActividadRESUMEN
We have developed a first generation of hybrid sparsomycin-linezolid compounds into a new family of orally bioavailable biaryloxazolidinones that have activity against both linezolid-susceptible and -resistant gram-positive bacteria as well as the fastidious gram-negative bacteria Haemophilus influenzae and Moraxella catarrahalis. The convergent synthesis of these new compounds is detailed.
Asunto(s)
Antibacterianos/síntesis química , Antibacterianos/farmacología , Oxazolidinonas/síntesis química , Oxazolidinonas/farmacología , Acetamidas/farmacología , Administración Oral , Antibacterianos/química , Cristalografía por Rayos X , Diseño de Fármacos , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Haemophilus influenzae/efectos de los fármacos , Linezolid , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Moraxella catarrhalis/efectos de los fármacos , Oxazolidinonas/química , Esparsomicina/farmacología , Relación Estructura-ActividadRESUMEN
Translocation is an essential step in the elongation cycle of the protein synthesis that allows for the continual incorporation of new amino acids to the growing polypeptide. Movement of mRNA and tRNAs within the ribosome is catalyzed by EF-G binding and GTP hydrolysis. The 30S subunit decoding center is crucial for the selection of the cognate tRNA. However, it is not clear whether the decoding center participates in translocation. We disrupted the interactions in the decoding center by mutating the universally conserved 16S rRNA bases G530, A1492, and A1493, and the effects of these mutations on translocation were studied. Our results show that point mutation of any of these 16S rRNA bases inhibits EF-G-dependent translocation. Furthermore, the mutant ribosomes showed increased puromycin reactivity in the pretranslocation complexes, indicating that the dynamic equilibrium of the peptidyl tRNA between the classical and hybrid-state configurations is influenced by contacts in the decoding center.
Asunto(s)
Extensión de la Cadena Peptídica de Translación/fisiología , Factor G de Elongación Peptídica/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , Guanosina Trifosfato/metabolismo , Hidrólisis , Mutagénesis Sitio-Dirigida , Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Mutación Puntual , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/fisiología , ARN Mensajero/metabolismo , ARN Ribosómico 16S/química , ARN Ribosómico 16S/genética , ARN de Transferencia/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/fisiología , Ribosomas/efectos de los fármacos , Ribosomas/fisiología , Esparsomicina/farmacología , Espectrometría de FluorescenciaRESUMEN
In Escherichia coli, interactions between the nascent TnaC-tRNA(Pro) peptidyl-tRNA and the translating ribosome create a tryptophan binding site in the ribosome where bound tryptophan inhibits TnaC-tRNA(Pro) cleavage. This inhibition delays ribosome release, thereby inhibiting Rho factor binding and action, resulting in increased tna operon transcription. Replacing Trp12 of TnaC with any other amino acid residue was previously shown to prevent tryptophan binding and induction of tna operon expression. Genome-wide comparisons of TnaC amino acid sequences identify Asp16 and Pro24, as well as Trp12, as highly conserved TnaC residues. Replacing these residues with other residues was previously shown to influence tryptophan induction of tna operon expression. In this study, in vitro analyses were performed to examine the potential roles of Asp16 and Pro24 in tna operon induction. Replacing Asp16 or Pro24 of TnaC of E. coli with other amino acids established that these residues are essential for free tryptophan binding and inhibition of TnaC-tRNA(Pro) cleavage at the peptidyl transferase center. Asp16 and Pro24 are in fact located in spatial positions corresponding to critical residues of AAP, another ribosome regulatory peptide. Sparsomycin-methylation protection studies further suggested that segments of 23S RNA were arranged differently in ribosomes bearing TnaCs with either the Asp16Ala or the Pro24Ala change. Thus, features of the amino acid sequence of TnaC of the nascent TnaC-tRNA(Pro) peptidyl-tRNA, in addition to the presence of Trp12, are necessary for the nascent peptide to create a tryptophan binding/inhibition site in the translating ribosome.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiología , Regulación Bacteriana de la Expresión Génica , Aminoacil-ARN de Transferencia/metabolismo , ARN de Transferencia de Prolina/metabolismo , Ribosomas/metabolismo , Triptófano/metabolismo , Sustitución de Aminoácidos/genética , Asparagina/genética , Secuencia Conservada , Proteínas de Escherichia coli/genética , Orden Génico , Mutagénesis Sitio-Dirigida , Operón , Prolina/genética , Inhibidores de la Síntesis de la Proteína/farmacología , Puromicina/farmacología , Aminoacil-ARN de Transferencia/genética , Factor Rho/metabolismo , Homología de Secuencia de Aminoácido , Esparsomicina/farmacologíaRESUMEN
To ensure accurate and rapid protein synthesis, nearby and distantly located functional regions of the ribosome must dynamically communicate and coordinate with one another through a series of information exchange networks. The ribosome is approximately 2/3 rRNA and information should pass mostly through this medium. Here, two viable mutants located in the peptidyltransferase center (PTC) of yeast ribosomes were created using a yeast genetic system that enables stable production of ribosomes containing only mutant rRNAs. The specific mutants were C2820U (Escherichia coli C2452) and Psi2922C (E. coli U2554). Biochemical and genetic analyses of these mutants suggest that they may trap the PTC in the 'open' or aa-tRNA bound conformation, decreasing peptidyl-tRNA binding. We suggest that these structural changes are manifested at the biological level by affecting large ribosomal subunit biogenesis, ribosomal subunit joining during initiation, susceptibility/resistance to peptidyltransferase inhibitors, and the ability of ribosomes to properly decode termination codons. These studies also add to our understanding of how information is transmitted both locally and over long distances through allosteric networks of rRNA-rRNA and rRNA-protein interactions.
Asunto(s)
Peptidil Transferasas/antagonistas & inhibidores , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Ribosómico/química , Ribosomas/química , Regulación Alostérica , Anisomicina/farmacología , Secuencia de Bases , Codón de Terminación , Resistencia a Medicamentos , Datos de Secuencia Molecular , Mutación , Paromomicina/farmacología , Peptidil Transferasas/metabolismo , Priones/metabolismo , ARN Ribosómico/genética , ARN de Transferencia/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Esparsomicina/farmacología , Levaduras/enzimología , Levaduras/genéticaRESUMEN
Here we report that sparsomycin, a streptococcal metabolite, enhances the replication of HIV-1 in multiple human T cell lines at a concentration of 400 nM. In addition to wild-type HIV-1, sparsomycin also accelerated the replication of low-fitness, drug-resistant mutants carrying either D30N or L90M within HIV-1 protease, which are frequently found mutations in HIV-1-infected patients on highly active antiretroviral therapy (HAART). Of particular interest was that replication enhancement appeared profound when HIV-1 such as the L90M-carrying mutant displayed relatively slower replication kinetics. The presence of sparsomycin did not immediately select the fast-replicating HIV-1 mutants in culture. In addition, sparsomycin did not alter the 50% inhibitory concentration (IC50) of antiretroviral drugs directed against HIV-1 including nucleoside reverse transcriptase inhibitors (lamivudine and stavudine), non-nucleoside reverse transcriptase inhibitor (nevirapine) and protease inhibitors (nelfinavir, amprenavir and indinavir). The IC50s of both zidovudine and lopinavir against multidrug resistant HIV-1 in the presence of sparsomycin were similar to those in the absence of sparsomycin. The frameshift reporter assay and Western blot analysis revealed that the replication-boosting effect was partly due to the sparsomycin's ability to increase the -1 frameshift efficiency required to produce the Gag-Pol transcript. In conclusion, the use of sparsomycin should be able to facilitate the drug resistance profiling of the clinical isolates and the study on the low-fitness viruses.
Asunto(s)
Farmacorresistencia Viral/efectos de los fármacos , VIH-1/efectos de los fármacos , Esparsomicina/farmacología , Replicación Viral/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , VIH-1/genética , Humanos , Concentración 50 Inhibidora , Modelos Biológicos , Mutación , Streptococcaceae/metabolismo , TransfecciónRESUMEN
The GTPase elongation factor (EF)-G is responsible for promoting the translocation of the messenger RNA-transfer RNA complex on the ribosome, thus opening up the A site for the next aminoacyl-tRNA. Chemical modification and cryo-EM studies have indicated that tRNAs can bind the ribosome in an alternative 'hybrid' state after peptidyl transfer and before translocation, though the relevance of this state during translation elongation has been a subject of debate. Here, using pre-steady-state kinetic approaches and mutant analysis, we show that translocation by EF-G is most efficient when tRNAs are bound in a hybrid state, supporting the argument that this state is an authentic intermediate during translation.
Asunto(s)
Extensión de la Cadena Peptídica de Translación , ARN de Transferencia/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Mutación/genética , Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Factor G de Elongación Peptídica/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN de Transferencia/genética , ARN de Transferencia de Metionina/genética , ARN de Transferencia de Metionina/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Esparsomicina/farmacologíaRESUMEN
Early studies demonstrated roles for ribosomal protein L3 in peptidyltransferase center formation and the ability of cells to propagate viruses. More recent studies have linked these two processes via the effects of mutants and drugs on programmed -1 ribosomal frameshifting. Here, we show that mutant forms of L3 result in ribosomes having increased affinities for both aminoacyl- and peptidyl-tRNAs. These defects potentiate the effects of sparsomycin, which promotes increased aminoalcyl-tRNA binding at the P-site, while antagonizing the effects anisomycin, a drug that promotes decreased peptidyl-tRNA binding at the A-site. The changes in ribosome affinities for tRNAs also correlate with decreased peptidyltransferase activities of mutant ribosomes, and with decreased rates of cell growth and protein synthesis. In vivo dimethylsulfate (DMS) protection studies reveal that small changes in L3 primary sequence also have significant effects on rRNA structure as far away as 100 A, supporting an allosteric model of ribosome function.
Asunto(s)
Regulación de la Expresión Génica , Haloarcula marismortui/metabolismo , Proteínas Ribosómicas/fisiología , Ribosomas/química , Sitio Alostérico , Anisomicina/farmacología , Secuencia de Bases , Cinética , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Estabilidad del ARN , ARN Ribosómico/química , Proteína Ribosomal L3 , Proteínas Ribosómicas/metabolismo , Esparsomicina/farmacología , Ésteres del Ácido Sulfúrico/farmacologíaRESUMEN
During protein synthesis, transfer RNAs (tRNAs) are translocated from the aminoacyl to peptidyl to exit sites of the ribosome, coupled to the movement of messenger RNA (mRNA), in a reaction catalyzed by elongation factor G (EF-G) and guanosine triphosphate (GTP). Here, we show that the peptidyl transferase inhibitor sparsomycin triggers accurate translocation in vitro in the absence of EF-G and GTP. Our results provide evidence that translocation is a function inherent to the ribosome and that the energy to drive this process is stored in the tRNA-mRNA-ribosome complex after peptide-bond formation. These findings directly implicate the peptidyl transferase center of the 50S subunit in the mechanism of translocation, a process involving large-scale movement of tRNA and mRNA in the 30S subunit, some 70 angstroms away.
Asunto(s)
ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Esparsomicina/farmacología , Acilación , Antibacterianos/farmacología , Catálisis , Codón , Inhibidores Enzimáticos/farmacología , Escherichia coli , Proteínas de Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli/metabolismo , Factor G de Elongación Peptídica/metabolismo , Peptidil Transferasas/antagonistas & inhibidores , Biosíntesis de Proteínas , ARN Bacteriano/efectos de los fármacos , ARN Bacteriano/metabolismo , ARN Mensajero/efectos de los fármacos , ARN de Transferencia/efectos de los fármacos , Ribosomas/efectos de los fármacosRESUMEN
Kinetic analysis of ribosomal peptidyltransferase activity in a methanolic puromycin reaction with wild type and drug-resistant 23 S RNA mutants was used to probe the structural basis of catalysis and mechanism of resistance to antibiotics. 23 S RNA mutants G2032A and G2447A are resistant to oxazolidinones both in vitro and in vivo with the latter displaying a 5-fold increase in the value of Km for initiator tRNA and a 100-fold decrease in Vmax in puromycin reaction. Comparison of the Ki values for oxazolidinones, chloramphenicol, and sparsomycin revealed partial cross-resistance between oxazolidinones and chloramphenicol; no cross-resistance was observed with sparsomycin, a known inhibitor of the peptidyltransferase A-site. Inhibition of the mutants using a truncated CCA-Phe-X-Biotin fragment as a P-site substrate is similar to that observed with the intact initiator tRNA, indicating that the inhibition is substrate-independent and that the peptidyltransferase itself is the oxazolidinone target. Mapping of all known mutations that confer resistance to these drugs onto the spatial structure of the 50 S ribosomal subunit allows for docking of an oxazolidinone into a proposed binding pocket. The model suggests that oxazolidinones bind between the P- and A-loops, partially overlapping with the peptidyltransferase P-site. Thus, kinetic, mutagenesis, and structural data suggest that oxazolidinones interfere with initiator fMet-tRNA binding to the P-site of the ribosomal peptidyltransferase center.
Asunto(s)
Oxazolidinonas/metabolismo , ARN Ribosómico 23S/genética , Antibióticos Antineoplásicos/farmacología , Sitios de Unión , Catálisis , Dominio Catalítico , Cloranfenicol/farmacología , Relación Dosis-Respuesta a Droga , Resistencia a Medicamentos , Escherichia coli/metabolismo , Concentración 50 Inhibidora , Cinética , Modelos Químicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Peptidil Transferasas/metabolismo , Unión Proteica , Inhibidores de la Síntesis de la Proteína/farmacología , Puromicina/farmacología , ARN de Transferencia de Metionina/metabolismo , Esparsomicina/farmacologíaRESUMEN
Over the last decade the yeast Saccharomyces cerevisiae has become a popular organism for studying heterologous gene expression and in vivo protein-protein interactions. Many variations of these basic systems have originated over the years. Besides these vast and varied applications of the yeast expression system, S. cerevisiae has also been used extensively in fundamental research as a model simple eukaryote. We have used the S. cerevisiae system to design a high throughput screen for anti-viral agents from natural sources. The design of the assay rests on the ability of the L-A helper virus and the M(1) satellite virus to detect small variations in -1 ribosomal frameshifting. A minor change in frameshifting efficiencies can be detected and clearly shown phenotypically in terms of zones of clearing on an agar plate. Using such a process, we have initiated a high throughput screening process for natural anti-viral agents.
Asunto(s)
Antivirales/farmacología , Evaluación Preclínica de Medicamentos/métodos , Sistema de Lectura Ribosómico , ARN/genética , Saccharomyces cerevisiae/fisiología , Anisomicina/farmacología , Cicloheximida/farmacología , Virus Helper/efectos de los fármacos , Virus Helper/fisiología , Humanos , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , ARN/metabolismo , Virus ARN/genética , Virus ARN/metabolismo , Virus Satélites/efectos de los fármacos , Virus Satélites/fisiología , Esparsomicina/farmacología , Replicación Viral/fisiologíaRESUMEN
Facile syntheses of sparsomycin (3) and its four analogues (4-7) based on diastereoselective oxidation of sulfide, sulfenylation, and coupling of 6-methyluracylacryllic acid with monooxodithioacetal amine, are described. Studies on the biological activity of morphological reversion on src(ts)-NRK cells were also carried out.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Transformación Celular Neoplásica/efectos de los fármacos , Esparsomicina/farmacología , Animales , Antibióticos Antineoplásicos/síntesis química , Virus del Sarcoma Aviar/metabolismo , Ratones , Oxidación-Reducción , Esparsomicina/síntesis química , Células Tumorales CultivadasRESUMEN
The antitumor antibiotic sparsomycin, produced by Streptomyces sparsogenes, is a universal translation inhibitor that blocks the peptide bond formation in ribosomes from all species. Sparsomycin-resistant strains were selected by transforming the sensitive Streptomyces lividans with an S. sparsogenes library. Resistance was linked to the presence of a plasmid containing an S. sparsogenes 5.9-kbp DNA insert. A restriction analysis of the insert traced down the resistance to a 3.6-kbp DNA fragment, which was sequenced. The analysis of the fragment nucleotide sequence together with the previous restriction data associate the resistance to srd, an open reading frame of 1,800 nucleotides. Ribosomes from S. sparsogenes and the S. lividans-resistant strains are equally sensitive to the inhibitor and bind the drug with similar affinity. Moreover, the drug was not modified by the resistant strains. However, resistant cells accumulated less antibiotic than the sensitive ones. In addition, membrane fractions from the resistant strains showed a higher capacity for binding the drug. The results indicate that resistance in the producer strain is not connected to either ribosome modification or drug inactivation, but it might be related to an alteration in the sparsomycin permeability barrier.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Esparsomicina/farmacología , Streptomyces/efectos de los fármacos , Antibióticos Antineoplásicos/metabolismo , Medios de Cultivo , ADN Bacteriano/genética , Resistencia a Medicamentos , Biblioteca Genómica , Cinética , Pruebas de Sensibilidad Microbiana , Fenilalanina/biosíntesis , Ribosomas/genética , Ribosomas/metabolismo , Esparsomicina/metabolismo , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismoRESUMEN
Our previous demonstration that mutants of 5S rRNA called mof9 can specifically alter efficiencies of programmed ribosomal frameshifting (PRF) suggested a role for this ubiquitous molecule in the maintenance of translational reading frame, though the repetitive nature of the 5S rDNA gene (>100 copies/cell) inhibited more detailed analyses. However, given the known interactions between 5S rRNA and ribosomal protein L5 (previously called L1 or YL3) encoded by an essential, single-copy gene, we monitored the effects of a series of well-defined rpl5 mutants on PRF and virus propagation. Consistent with the mof9 results, we find that the rpl5 mutants promoted increased frameshifting efficiencies in both the -1 and +1 directions, and conferred defects in the ability of cells to propagate two endogenous viruses. Biochemical analyses demonstrated that mutant ribosomes had decreased affinities for peptidyl-tRNA. Pharmacological studies showed that sparsomycin, a peptidyltransferase inhibitor that specifically increases the binding of peptidyl-tRNA with ribosomes, was antagonistic to the frameshifting defects of the most severe mutant, and the extent of sparsomycin resistance correlated with the severity of the frameshifting defects in all of the mutants. These results provide biochemical and physiological evidence that one function of L5 is to anchor peptidyl-tRNA to the P-site. A model is presented describing how decreased affinity of ribosomes for peptidyl-tRNA can affect both -1 and +1 frameshifting, and for the effects of sparsomycin.
Asunto(s)
ARN de Transferencia/metabolismo , Proteínas Ribosómicas/química , Saccharomyces cerevisiae/metabolismo , Alelos , Anisomicina/farmacología , Antibióticos Antineoplásicos/farmacología , Relación Dosis-Respuesta a Droga , Mutación del Sistema de Lectura , Mutación , Peptidil Transferasas/antagonistas & inhibidores , Fenotipo , Plásmidos/metabolismo , Biosíntesis de Proteínas , Inhibidores de la Síntesis de la Proteína/farmacología , Retroelementos/genética , Ribosomas/metabolismo , Esparsomicina/farmacología , Factores de TiempoRESUMEN
The antitumor antibiotic sparsomycin is a universal and potent inhibitor of peptide bond formation and selectively acts on several human tumors. It binds to the ribosome strongly, at an unknown site, in the presence of an N-blocked donor tRNA substrate, which it stabilizes on the ribosome. Its site of action was investigated by inducing a crosslink between sparsomycin and bacterial, archaeal, and eukaryotic ribosomes complexed with P-site-bound tRNA, on irradiating with low energy ultraviolet light (at 365 nm). The crosslink was localized exclusively to the universally conserved nucleotide A2602 within the peptidyl transferase loop region of 23S-like rRNA by using a combination of a primer extension approach, RNase H fragment analysis, and crosslinking with radioactive [(125)I]phenol-alanine-sparsomycin. Crosslinking of several sparsomycin derivatives, modified near the sulfoxy group, implicated the modified uracil residue in the rRNA crosslink. The yield of the antibiotic crosslink was weak in the presence of deacylated tRNA and strong in the presence of an N-blocked P-site-bound tRNA, which, as was shown earlier, increases the accessibility of A2602 on the ribosome. We infer that both A2602 and its induced conformational switch are critically important both for the peptidyl transfer reaction and for antibiotic inhibition. This supposition is reinforced by the observation that other antibiotics that can prevent peptide bond formation in vitro inhibit, to different degrees, formation of the crosslink.
Asunto(s)
Antibióticos Antineoplásicos/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Escherichia coli/metabolismo , Peptidil Transferasas/metabolismo , ARN Ribosómico 23S/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Esparsomicina/análogos & derivados , Esparsomicina/metabolismo , Antibióticos Antineoplásicos/farmacología , Bacillus megaterium/metabolismo , Secuencia de Bases , Reactivos de Enlaces Cruzados/farmacología , Halobacterium salinarum/metabolismo , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Peptidil Transferasas/química , ARN Bacteriano/metabolismo , ARN de Hongos/metabolismo , ARN Ribosómico 23S/química , ARN de Transferencia/química , Ribosomas/efectos de los fármacos , Ribosomas/ultraestructura , Saccharomyces cerevisiae/metabolismo , Esparsomicina/farmacologíaRESUMEN
It was recently demonstrated that peptide bond formation can occur using an Escherichia coli naked 23S ribosomal RNA without any of the ribosomal proteins. Here, the six domains of the 23S ribosomal RNA were individually synthesized and shown to be capable, when complexed together, of stimulating the reaction. Omission and addition experiments indicated that the activity could be reconstituted solely by domain V at a concentration 10 times higher than that of the intact 23S ribosomal RNA, whereas domain VI could enhance the activity in trans. These findings suggest that fragments of an RNA molecule have the ability to associate into a functional whole.
Asunto(s)
Escherichia coli/metabolismo , Biosíntesis de Péptidos , Peptidil Transferasas/metabolismo , ARN Ribosómico 23S/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , Catálisis , Neomicina/farmacología , Conformación de Ácido Nucleico , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN Ribosómico 23S/química , Esparsomicina/farmacología , Transcripción GenéticaRESUMEN
A detailed kinetic study was carried out on the inhibitory mechanisms of two eukaryotic peptidyltransferase drugs (I), anisomycin and sparsomycin. In an in vitro system from rabbit reticulocytes, AcPhe-puromycin is produced in a pseudo-first-order reaction from the preformed AcPhe-tRNA/poly(U)/80S ribosome complex (complex C) and excess puromycin (S). This reaction is inhibited by anisomycin and sparsomycin through different mechanisms. Anisomycin acts as a mixed noncompetitive inhibitor. The product, AcPhe-puromycin, is derived only from C according to the puromycin reaction. On the other hand, sparsomycin reacts with complex C in a two-step reaction, [REACTION; SEE TEXT] An initial rapid binding of the drug produces the encounter complex CI. During this step and before conversion of CI to C*I, sparsomycin behaves as a competitive inhibitor. The rapidly produced CI is isomerized slowly to a conformationally altered species C*I in which I is bound more tightly. The rate constants of this step are k6 = 2.1 min-1 and k7 = 0.095 min-1. Moreover, the low value of the association rate constant k7/Ki' (2 x 10(5) M-1 sec-1), provides insight into the rates of possible conformational changes occurring during protein synthesis and supports the proposal that sparsomycin is the first example of a slow-binding inhibitor of eukaryotic peptidyltransferase. When complex C is preincubated with concentrations of sparsomycin of >8 Ki and then reacts with a mixture of puromycin and sparsomycin, the inhibition becomes linear mixed noncompetitive and involves C*I instead of CI. During this phase, AcPhe-puromycin is produced from a new, modified ribosomal complex with a lower catalytic rate constant. Thus, sparsomycin also acts as a modifier of eukaryotic peptidyltransferase activity.
Asunto(s)
Anisomicina/farmacología , Peptidil Transferasas/antagonistas & inhibidores , Inhibidores de la Síntesis de la Proteína/farmacología , Reticulocitos/enzimología , Esparsomicina/farmacología , Animales , Cinética , Puromicina/metabolismo , ConejosRESUMEN
Three pyrimidinylpropanamide antibiotics sparsomycin (1), sparoxomycins A1, A2 (2, 3), and also six analogues (4-9) have been synthesized by employing asymmetric sulfide oxidation conditions as a key step. Sparsomycin (1) and its alkyl analogues (5-7) showed higher morphological reversion activities on srctsNRK cells than 2 and 3.