RESUMO
Screening for inhibitors of bacterial protein synthesis Initiation Factor 2 (IF2) binding to N-formyl-Methionyl-transfer RNA (fMet-tRNA((fMet))) identified a series of aminoglycosides, that included amikacin and kanamycin A1, as inhibitors of this interaction. Subsequent testing revealed that aminoglycosides displayed a wide range of inhibitory activity. However, the failure of these compounds to completely inhibit binding of IF2 to fMet-tRNA((fMet)), the known ability of aminoglycosides to bind RNA, and the ability of the aminoglycosides to displace PicoGreen bound to fMet-tRNA((fMet)) suggest these compounds act by binding fMet-tRNA((fMet)). This hypothesis is further supported by isothermal denaturation experiments that failed to show any interaction between the IF2 protein and the aminoglycosides.
Assuntos
Antibacterianos/farmacologia , Bactérias/metabolismo , Fator de Iniciação 2 em Procariotos/metabolismo , RNA de Transferência de Metionina/metabolismo , Amicacina/farmacologia , Antibacterianos/química , Bactérias/efeitos dos fármacos , Sequência de Carboidratos , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Canamicina/farmacologia , Dados de Sequência Molecular , Fator de Iniciação 2 em Procariotos/efeitos dos fármacos , Desnaturação Proteica , RNA de Transferência de Metionina/efeitos dos fármacosRESUMO
The oxazolidinones are a novel class of antimicrobial agents that target protein synthesis in a wide spectrum of gram-positive and anaerobic bacteria. The oxazolidinone PNU-100766 (linezolid) inhibits the binding of fMet-tRNA to 70S ribosomes. Mutations to oxazolidinone resistance in Halobacterium halobium, Staphylococcus aureus, and Escherichia coli map at or near domain V of the 23S rRNA, suggesting that the oxazolidinones may target the peptidyl transferase region responsible for binding fMet-tRNA. This study demonstrates that the potency of oxazolidinones corresponds to increased inhibition of fMet-tRNA binding. The inhibition of fMet-tRNA binding is competitive with respect to the fMet-tRNA concentration, suggesting that the P site is affected. The fMet-tRNA reacts with puromycin to form peptide bonds in the presence of elongation factor P (EF-P), which is needed for optimum specificity and efficiency of peptide bond synthesis. Oxazolidinone inhibition of the P site was evaluated by first binding fMet-tRNA to the A site, followed by translocation to the P site with EF-G. All three of the oxazolidinones used in this study inhibited translocation of fMet-tRNA. We propose that the oxazolidinones target the ribosomal P site and pleiotropically affect fMet-tRNA binding, EF-P stimulated synthesis of peptide bonds, and, most markedly, EF-G-mediated translocation of fMet-tRNA into the P site.
Assuntos
Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Oxazóis/farmacologia , Fatores de Alongamento de Peptídeos/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Acetamidas/farmacologia , Proteínas de Bactérias/biossíntese , Translocação Bacteriana/genética , Escherichia coli/ultraestrutura , Cinética , Linezolida , Oxazolidinonas/farmacologia , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Fator G para Elongação de Peptídeos/genética , Fatores de Alongamento de Peptídeos/genética , Peptidil Transferases/química , Biossíntese de Proteínas/genética , RNA de Transferência de Metionina/efeitos dos fármacos , Ribossomos/químicaRESUMO
Ricin A chain caused inhibition of protein synthesis by reticulocyte lysate with concomitant depurination of 28S rRNA. The partial reaction(s) of protein synthesis inhibited was investigated by following the appearance of [35S]methionine from initiator [35S]Met-tRNA into 40S ribosomal subunits, 80S monosomes and polysomes. Ricin A chain caused an accumulation of [35S]Met in monosomes which did not enter polysomes. In these respects the effects of the ricin A chain resembled those of diphtheria toxin, an inhibitor of elongation-factor-2-catalyzed translocation. This is consistent with the previously proposed site of action of ricin as an inhibitor of elongation. However, the inhibitory effects of the ricin A chain and diphtheria toxin are not equivalent because we observed that the rate of formation of the 80S initiation complex was reduced approximately sixfold with the ricin A chain relative to diphtheria toxin. Analysis of methionine-containing peptides bound to 80S monosomes in ricin-A-chain-inhibited and diphtheria-toxin-inhibited lysates, programmed with globin mRNA, revealed a predominance of Met-Val, suggesting that the elongation cycle is inhibited at the translocation step. Translocation was also implicated as the step blocked in both the ricin-A-chain-inhibited and diphtheria-toxin-inhibited lysates, by the finding that nascent peptide chains were unreactive towards puromycin. It is concluded that ricin-A-chain-modified ribosomes are deficient in two protein synthesis partial reactions: the formation of the 80S initiation complex during initiation and the translocation step of the elongation cycle.
