RESUMEN
Aminoglycosides are widely used antibiotics that cause messenger RNA decoding errors, block mRNA and transfer RNA translocation, and inhibit ribosome recycling. Ribosome recycling follows the termination of protein synthesis and is aided by ribosome recycling factor (RRF) in bacteria. The molecular mechanism by which aminoglycosides inhibit ribosome recycling is unknown. Here we show in X-ray crystal structures of the Escherichia coli 70S ribosome that RRF binding causes RNA helix H69 of the large ribosomal subunit, which is crucial for subunit association, to swing away from the subunit interface. Aminoglycosides bind to H69 and completely restore the contacts between ribosomal subunits that are disrupted by RRF. These results provide a structural explanation for aminoglycoside inhibition of ribosome recycling.
Asunto(s)
Aminoglicósidos/química , Antibacterianos/química , Escherichia coli/efectos de los fármacos , Proteínas Ribosómicas/química , Ribosomas/química , Aminoglicósidos/farmacología , Antibacterianos/farmacología , Sitios de Unión , Cristalografía por Rayos X , Escherichia coli/genética , Gentamicinas/química , Gentamicinas/farmacología , Modelos Moleculares , Estructura Molecular , Neomicina/química , Neomicina/farmacología , Paromomicina/química , Paromomicina/farmacología , Subunidades de Proteína/química , Relación Estructura-ActividadRESUMEN
The prokaryotic ribosome is an important target of antibiotic action. We determined the X-ray structure of the aminoglycoside kasugamycin (Ksg) in complex with the Escherichia coli 70S ribosome at 3.5-A resolution. The structure reveals that the drug binds within the messenger RNA channel of the 30S subunit between the universally conserved G926 and A794 nucleotides in 16S ribosomal RNA, which are sites of Ksg resistance. To our surprise, Ksg resistance mutations do not inhibit binding of the drug to the ribosome. The present structural and biochemical results indicate that inhibition by Ksg and Ksg resistance are closely linked to the structure of the mRNA at the junction of the peptidyl-tRNA and exit-tRNA sites (P and E sites).
Asunto(s)
Aminoglicósidos/farmacología , Antibacterianos/farmacología , Escherichia coli/química , Biosíntesis de Proteínas , ARN Bacteriano/química , ARN Mensajero/química , Aminoglicósidos/química , Aminoglicósidos/metabolismo , Antibacterianos/química , Secuencia de Bases , Sitios de Unión , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína , Ribosomas/genética , Ribosomas/metabolismo , Relación Estructura-Actividad , Moldes GenéticosRESUMEN
During environmental stress, organisms limit protein synthesis by storing inactive ribosomes that are rapidly reactivated when conditions improve. Here we present structural and biochemical data showing that protein Y, an Escherichia coli stress protein, fills the tRNA- and mRNA-binding channel of the small ribosomal subunit to stabilize intact ribosomes. Protein Y inhibits translation initiation during cold shock but not at normal temperatures. Furthermore, protein Y competes with conserved translation initiation factors that, in bacteria, are required for ribosomal subunit dissociation. The mechanism used by protein Y to reduce translation initiation during stress and quickly release ribosomes for renewed translation initiation may therefore occur widely in nature.
Asunto(s)
Biosíntesis de Proteínas , Proteínas Ribosómicas/química , Proteínas Ribosómicas/fisiología , Proteínas Bacterianas , Sitios de Unión , Cristalografía por Rayos X , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Modelos Biológicos , Modelos Moleculares , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/química , Ribosomas/metabolismo , Ribosomas/ultraestructura , Temperatura , Rayos XRESUMEN
We have characterised the interaction of the Aeropyrum pernix origin recognition complex proteins (ORC1 and ORC2) with DNA using DNase I footprinting. Each protein binds upstream of its respective gene. However, ORC1 protein alone interacts more tightly with an additional region containing multiple origin recognition box (ORB) sites that we show to be a replication origin. At this origin, there are four ORB elements disposed either side of an A+T-rich region. An ORC1 protein dimer binds at each of these ORB sites. Once all four ORB sites have bound ORC1 protein, there is a transition to a higher-order assembly with a defined alteration in topology and superhelicity. Furthermore, after this transition, the A+T-rich region becomes sensitive to digestion by DNase I and P1 nuclease, revealing that the transition promotes distortion of the DNA in this region, presumably as a prelude to loading of MCM helicase.
Asunto(s)
Aeropyrum , Proteínas Arqueales/metabolismo , Complejo de Reconocimiento del Origen/metabolismo , Origen de Réplica , Aeropyrum/genética , Aeropyrum/metabolismo , Proteínas Arqueales/genética , Secuencia de Bases , ADN/química , ADN/metabolismo , Huella de ADN , Radical Hidroxilo/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Complejo de Reconocimiento del Origen/genética , Unión Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismoRESUMEN
At the end of translation in bacteria, ribosome recycling factor (RRF) is used together with elongation factor G to recycle the 30S and 50S ribosomal subunits for the next round of translation. In x-ray crystal structures of RRF with the Escherichia coli 70S ribosome, RRF binds to the large ribosomal subunit in the cleft that contains the peptidyl transferase center. Upon binding of either E. coli or Thermus thermophilus RRF to the E. coli ribosome, the tip of ribosomal RNA helix 69 in the large subunit moves away from the small subunit toward RRF by 8 A, thereby disrupting a key contact between the small and large ribosomal subunits termed bridge B2a. In the ribosome crystals, the ability of RRF to destabilize bridge B2a is influenced by crystal packing forces. Movement of helix 69 involves an ordered-to-disordered transition upon binding of RRF to the ribosome. The disruption of bridge B2a upon RRF binding to the ribosome seen in the present structures reveals one of the key roles that RRF plays in ribosome recycling, the dissociation of 70S ribosomes into subunits. The structures also reveal contacts between domain II of RRF and protein S12 in the 30S subunit that may also play a role in ribosome recycling.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Ribosomas/química , Ribosomas/metabolismo , Cristalografía por Rayos X , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Estructura Terciaria de Proteína , ARN Ribosómico/química , ARN Ribosómico/metabolismo , Thermus thermophilus/química , Thermus thermophilus/metabolismoRESUMEN
DNA replication in archaea and in eukaryotes share many similarities. We report the structure of an archaeal origin recognition complex protein, ORC1, bound to an origin recognition box, a DNA sequence that is found in multiple copies at replication origins. DNA binding is mediated principally by a C-terminal winged helix domain that inserts deeply into the major and minor grooves, widening them both. However, additional DNA contacts are made with the N-terminal AAA+ domain, which inserts into the minor groove at a characteristic G-rich sequence, inducing a 35 degrees bend in the duplex and providing directionality to the binding site. Both contact regions also induce substantial unwinding of the DNA. The structure provides insight into the initial step in assembly of a replication origin and recruitment of minichromosome maintenance (MCM) helicase to that origin.
Asunto(s)
Aeropyrum/química , Proteínas Arqueales/química , ADN de Archaea/química , Complejo de Reconocimiento del Origen/química , Origen de Réplica , Aeropyrum/metabolismo , Sitios de Unión , Cristalografía por Rayos X , ADN de Archaea/metabolismo , Dimerización , Modelos Moleculares , Conformación de Ácido Nucleico , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
We describe two structures of the intact bacterial ribosome from Escherichia coli determined to a resolution of 3.5 angstroms by x-ray crystallography. These structures provide a detailed view of the interface between the small and large ribosomal subunits and the conformation of the peptidyl transferase center in the context of the intact ribosome. Differences between the two ribosomes reveal a high degree of flexibility between the head and the rest of the small subunit. Swiveling of the head of the small subunit observed in the present structures, coupled to the ratchet-like motion of the two subunits observed previously, suggests a mechanism for the final movements of messenger RNA (mRNA) and transfer RNAs (tRNAs) during translocation.