RESUMEN
Understanding the mechanisms of inhibitors of translation termination may inform development of new antibacterials and therapeutics for premature termination diseases. We report the crystal structure of the potent termination inhibitor blasticidin S bound to the ribosomal 70Sâ¢release factor 1 (RF1) termination complex. Blasticidin S shifts the catalytic domain 3 of RF1 and restructures the peptidyl transferase center. Universally conserved uridine 2585 in the peptidyl transferase center occludes the catalytic backbone of the GGQ motif of RF1, explaining the structural mechanism of inhibition. Rearrangement of domain 3 relative to the codon-recognition domain 2 provides insight into the dynamics of RF1 implicated in termination accuracy.
Asunto(s)
Antibacterianos/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Dominio Catalítico/efectos de los fármacos , Codón de Terminación/metabolismo , Modelos Moleculares , Nucleósidos/antagonistas & inhibidores , Terminación de la Cadena Péptídica Traduccional/efectos de los fármacos , Factores de Terminación de Péptidos/efectos de los fármacos , Peptidil Transferasas/metabolismo , Conformación Proteica , Ribosomas/efectos de los fármacos , Ribosomas/metabolismoRESUMEN
Listeria monocytogenes strains expressing high levels of the virulence regulator PrfA (mutant PrfA* or wild-type PrfA) show strong growth inhibition in minimal media when they are supplemented with glucose but not when they are supplemented with glucose-6-phosphate compared to the growth of isogenic strains expressing low levels of PrfA. A significantly reduced rate of glucose uptake was observed in a PrfA*-overexpressing strain growing in LB supplemented with glucose. Comparative transcriptome analyses were performed with RNA isolated from a prfA mutant and an isogenic strain carrying multiple copies of prfA or prfA* on a plasmid. These analyses revealed that in addition to high transcriptional up-regulation of the known PrfA-regulated virulence genes (group I), there was less pronounced up-regulation of the expression of several phage and metabolic genes (group II) and there was strong down-regulation of several genes involved mainly in carbon and nitrogen metabolism in the PrfA*-overexpressing strain (group III). Among the latter genes are the nrgAB, gltAB, and glnRA operons (involved in nitrogen metabolism), the ilvB operon (involved in biosynthesis of the branched-chain amino acids), and genes for some ABC transporters. Most of the down-regulated genes have been shown previously to belong to a class of genes in Bacillus subtilis whose expression is negatively affected by impaired glucose uptake. Our results lead to the conclusion that excess PrfA (or PrfA*) interferes with a component(s) essential for phosphotransferase system-mediated glucose transport.
Asunto(s)
Glucosa/farmacología , Listeria monocytogenes/crecimiento & desarrollo , Factores de Terminación de Péptidos/genética , Secuencia de Bases , Transporte Biológico , Cartilla de ADN , Glucosa/metabolismo , Cinética , Listeria monocytogenes/efectos de los fármacos , Listeria monocytogenes/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de Terminación de Péptidos/efectos de los fármacos , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/antagonistas & inhibidores , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , ARN Bacteriano , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Acetaldehyde is the first oxidation product of ethanol in vivo. Our earlier work showed that with sufficient acetaldehyde, five of the six possible sites of the peptide pentalysine were modified as a Schiff base (Braun KP, et al: J Biol Chem 270:11263-11266, 1995). However, we were unable to deduce unequivocally which site was unmodified. Lysine residues, as well as the amine terminal valine residues, in hemoglobin have been implicated as target structures for acetaldehyde adducts resulting from ethanol consumption. Hemoglobin adducts of acetaldehyde have been used clinically as a marker of ethanol consumption, but the chemical nature of these adducts remains undefined. As part of our continuing structural characterization of acetaldehyde-protein adduct formation, we studied the peptides Val-His-Leu-Thr-Pro and Val-His-Leu-Thr-Pro-Val-Glu-Lys, from the amine terminus of the beta-globin chain of hemoglobin, in vitro. Both peptides have at least one potential site for adduct formation. In the octapeptide, the N-terminal amine group of Val as well as the epsilon-amine group of the lysine sidechain can potentially be modified by acetaldehyde. We used mass spectrometry, carbon-13 nuclear magnetic resonance, and Raman spectroscopy and characterized stable Schiff base acetaldehyde adducts of these two peptides at both reactive sites. The identification of stable Schiff base adducts with the N-terminal peptides of the beta-chain of hemoglobin as well as with epsilon-amino groups of lysine provides another possible means of monitoring ethanol consumption. The functional implications of these stable Schiff bases remains undefined.