RESUMEN
During stress conditions such as heat shock and antibiotic exposure, ribosomes stall on messenger RNAs, leading to inhibition of protein synthesis. To remobilize ribosomes, bacteria use rescue factors such as HflXr, a homolog of the conserved housekeeping GTPase HflX that catalyzes the dissociation of translationally inactive ribosomes into individual subunits. Here we use time-resolved cryo-electron microscopy to elucidate the mechanism of ribosome recycling by Listeria monocytogenes HflXr. Within the 70S ribosome, HflXr displaces helix H69 of the 50S subunit and induces long-range movements of the platform domain of the 30S subunit, disrupting inter-subunit bridges B2b, B2c, B4, B7a and B7b. Our findings unveil a unique ribosome recycling strategy by HflXr which is distinct from that mediated by RRF and EF-G. The resemblance between HflXr and housekeeping HflX suggests that the alternative ribosome recycling mechanism reported here is universal in the prokaryotic kingdom.
Asunto(s)
Proteínas Bacterianas , Microscopía por Crioelectrón , Listeria monocytogenes , Ribosomas , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Ribosomas/metabolismo , Listeria monocytogenes/metabolismo , Listeria monocytogenes/genética , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/química , Biosíntesis de Proteínas , Modelos Moleculares , Subunidades Ribosómicas Grandes Bacterianas/metabolismo , Subunidades Ribosómicas Grandes Bacterianas/química , Subunidades Ribosómicas Grandes Bacterianas/ultraestructura , Factor G de Elongación Peptídica/metabolismo , Factor G de Elongación Peptídica/químicaRESUMEN
In all living cells, the ribosome translates the genetic information carried by messenger RNAs (mRNAs) into proteins. The process of ribosome recycling, a key step during protein synthesis that ensures ribosomal subunits remain available for new rounds of translation, has been largely overlooked. Despite being essential to the survival of the cell, several mechanistic aspects of ribosome recycling remain unclear. In eubacteria and mitochondria, recycling of the ribosome into subunits requires the concerted action of the ribosome recycling factor (RRF) and elongation factor G (EF-G). Recently, the conserved protein HflX was identified in bacteria as an alternative factor that recycles the ribosome under stress growth conditions. The homologue of HflX, the GTP-binding protein 6 (GTPBP6), has a dual role in mitochondrial translation by facilitating ribosome recycling and biogenesis. In this review, mechanisms of ribosome recycling in eubacteria and mitochondria are described based on structural studies of ribosome complexes.
Asunto(s)
Factor G de Elongación Peptídica , Ribosomas , Bacterias/genética , Bacterias/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Factor G de Elongación Peptídica/química , Factor G de Elongación Peptídica/genética , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Ribosomas/metabolismoRESUMEN
Aminoglycosides are a class of antibiotics that bind to ribosomal RNA and exert pleiotropic effects on ribosome function. Amikacin, the semisynthetic derivative of kanamycin, is commonly used for treating severe infections with multidrug-resistant, aerobic Gram-negative bacteria. Amikacin carries the 4-amino-2-hydroxy butyrate (AHB) moiety at the N1 amino group of the central 2-deoxystreptamine (2-DOS) ring, which may confer amikacin a unique ribosome inhibition profile. Here we use in vitro fast kinetics combined with X-ray crystallography and cryo-EM to dissect the mechanisms of ribosome inhibition by amikacin and the parent compound, kanamycin. Amikacin interferes with tRNA translocation, release factor-mediated peptidyl-tRNA hydrolysis, and ribosome recycling, traits attributed to the additional interactions amikacin makes with the decoding center. The binding site in the large ribosomal subunit proximal to the 3'-end of tRNA in the peptidyl (P) site lays the groundwork for rational design of amikacin derivatives with improved antibacterial properties.