RESUMEN
Translation termination involves release factors RF1, RF2 and the GTPase RF3 that recycles RF1 and RF2 from the ribosome. RF3 dissociates from the ribosome in the GDP-bound form and must then exchange GDP for GTP. The 70S ribosome termination complex (70S-TC) accelerates GDP exchange in RF3, suggesting that the 70S-TC can function as the guanine nucleotide exchange factor for RF3. Here, we use cryogenic-electron microscopy to elucidate the mechanism of GDP dissociation from RF3 catalyzed by the Escherichia coli 70S-TC. The non-rotated ribosome bound to RF1 remodels RF3 and induces a peptide flip in the phosphate-binding loop, efficiently ejecting GDP. Binding of GTP allows RF3 to dock at the GTPase center, promoting the dissociation of RF1 from the ribosome. The structures recapitulate the functional cycle of RF3 on the ribosome and uncover the mechanism by which the 70S-TC allosterically dismantles the phosphate-binding groove in RF3, a previously overlooked function of the ribosome.
RESUMEN
The frequency of errors upon decoding of messenger RNA by the bacterial ribosome is low, with one misreading event per 1 × 104 codons. In the universal genetic code, the AUN codon box specifies two amino acids, isoleucine and methionine. In bacteria and archaea, decoding specificity of the AUA and AUG codons relies on the wobble avoidance strategy that requires modification of C34 in the anticodon loop of isoleucine transfer RNAIleCAU (tRNAIleCAU). Bacterial tRNAIleCAU with 2-lysylcytidine (lysidine) at the wobble position deciphers AUA while avoiding AUG. Here we report cryo-electron microscopy structures of the Escherichia coli 70S ribosome complexed with elongation factor thermo unstable (EF-Tu) and isoleucine-tRNAIleLAU in the process of decoding AUA and AUG. Lysidine in tRNAIleLAU excludes AUG by promoting the formation of an unusual Hoogsteen purine-pyrimidine nucleobase geometry at the third position of the codon, weakening the interactions with the mRNA and destabilizing the EF-Tu ternary complex. Our findings elucidate the molecular mechanism by which tRNAIleLAU specifically decodes AUA over AUG.
Asunto(s)
Microscopía por Crioelectrón , Escherichia coli , Modelos Moleculares , Factor Tu de Elongación Peptídica , ARN de Transferencia de Isoleucina , Ribosomas , Factor Tu de Elongación Peptídica/metabolismo , Factor Tu de Elongación Peptídica/química , Factor Tu de Elongación Peptídica/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Ribosomas/metabolismo , Ribosomas/ultraestructura , Ribosomas/química , ARN de Transferencia de Isoleucina/metabolismo , ARN de Transferencia de Isoleucina/química , ARN de Transferencia de Isoleucina/genética , Codón/metabolismo , Codón/genética , Anticodón/química , Anticodón/metabolismo , Conformación de Ácido Nucleico , Isoleucina/metabolismo , Isoleucina/química , ARN Mensajero/metabolismo , ARN Mensajero/química , ARN Mensajero/genética , Lisina/análogos & derivados , Nucleósidos de PirimidinaRESUMEN
To conserve energy during starvation and stress, many organisms use hibernation factor proteins to inhibit protein synthesis and protect their ribosomes from damage1,2. In bacteria, two families of hibernation factors have been described, but the low conservation of these proteins and the huge diversity of species, habitats and environmental stressors have confounded their discovery3-6. Here, by combining cryogenic electron microscopy, genetics and biochemistry, we identify Balon, a new hibernation factor in the cold-adapted bacterium Psychrobacter urativorans. We show that Balon is a distant homologue of the archaeo-eukaryotic translation factor aeRF1 and is found in 20% of representative bacteria. During cold shock or stationary phase, Balon occupies the ribosomal A site in both vacant and actively translating ribosomes in complex with EF-Tu, highlighting an unexpected role for EF-Tu in the cellular stress response. Unlike typical A-site substrates, Balon binds to ribosomes in an mRNA-independent manner, initiating a new mode of ribosome hibernation that can commence while ribosomes are still engaged in protein synthesis. Our work suggests that Balon-EF-Tu-regulated ribosome hibernation is a ubiquitous bacterial stress-response mechanism, and we demonstrate that putative Balon homologues in Mycobacteria bind to ribosomes in a similar fashion. This finding calls for a revision of the current model of ribosome hibernation inferred from common model organisms and holds numerous implications for how we understand and study ribosome hibernation.
Asunto(s)
Proteínas Bacterianas , Respuesta al Choque por Frío , Factores de Terminación de Péptidos , Biosíntesis de Proteínas , Psychrobacter , Proteínas Ribosómicas , Ribosomas , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Factor Tu de Elongación Peptídica/química , Factor Tu de Elongación Peptídica/metabolismo , Factor Tu de Elongación Peptídica/ultraestructura , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/ultraestructura , Ribosomas/química , Ribosomas/metabolismo , Ribosomas/ultraestructura , Psychrobacter/química , Psychrobacter/genética , Psychrobacter/metabolismo , Psychrobacter/ultraestructura , Microscopía por Crioelectrón , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Factores de Terminación de Péptidos/ultraestructuraRESUMEN
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
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.
Asunto(s)
Amicacina , Antibacterianos , Amicacina/farmacología , Amicacina/química , Amicacina/metabolismo , Antibacterianos/química , Modelos Moleculares , Ribosomas/metabolismo , Kanamicina/farmacología , Kanamicina/análisis , Kanamicina/metabolismo , ARN de Transferencia/metabolismoRESUMEN
Thermorubin (THR) is an aromatic anthracenopyranone antibiotic active against both Gram-positive and Gram-negative bacteria. It is known to bind to the 70S ribosome at the intersubunit bridge B2a and was thought to inhibit factor-dependent initiation of translation and obstruct the accommodation of tRNAs into the A site. Here, we show that thermorubin causes ribosomes to stall in vivo and in vitro at internal and termination codons, thereby allowing the ribosome to initiate protein synthesis and translate at least a few codons before stalling. Our biochemical data show that THR affects multiple steps of translation elongation with a significant impact on the binding stability of the tRNA in the A site, explaining premature cessation of translation. Our high-resolution crystal and cryo-EM structures of the 70S-THR complex show that THR can co-exist with P- and A-site tRNAs, explaining how ribosomes can elongate in the presence of the drug. Remarkable is the ability of THR to arrest ribosomes at the stop codons. Our data suggest that by causing structural re-arrangements in the decoding center, THR interferes with the accommodation of tRNAs or release factors into the ribosomal A site.
Asunto(s)
Antraquinonas , Antibacterianos , Bacterias Gramnegativas , Bacterias Grampositivas , Biosíntesis de Proteínas , Antibacterianos/farmacología , Codón de Terminación/metabolismo , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Ribosomas/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Antraquinonas/farmacologíaRESUMEN
During translation initiation, initiation factor 2 (IF2) holds initiator transfer RNA (fMet-tRNAifMet) in a specific orientation in the peptidyl (P) site of the ribosome. Upon subunit joining IF2 hydrolyzes GTP and, concomitant with inorganic phosphate (Pi) release, changes conformation facilitating fMet-tRNAifMet accommodation into the P site and transition of the 70 S ribosome initiation complex (70S-IC) to an elongation-competent ribosome. The mechanism by which IF2 separates from initiator tRNA at the end of translation initiation remains elusive. Here, we report cryo-electron microscopy (cryo-EM) structures of the 70S-IC from Pseudomonas aeruginosa bound to compact IF2-GDP and initiator tRNA. Relative to GTP-bound IF2, rotation of the switch 2 α-helix in the G-domain bound to GDP unlocks a cascade of large-domain movements in IF2 that propagate to the distal tRNA-binding domain C2. The C2-domain relocates 35 angstroms away from tRNA, explaining how IF2 makes way for fMet-tRNAifMet accommodation into the P site. Our findings provide the basis by which IF2 gates the ribosome to the elongation phase.
Asunto(s)
Factor 2 Procariótico de Iniciación , ARN de Transferencia de Metionina , Microscopía por Crioelectrón , Guanosina Trifosfato/metabolismo , Factor 2 Procariótico de Iniciación/química , ARN de Transferencia de Metionina/metabolismo , Ribosomas/metabolismoRESUMEN
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
The bacterial ribosome is recycled into subunits by two conserved proteins, elongation factor G (EF-G) and the ribosome recycling factor (RRF). The molecular basis for ribosome recycling by RRF and EF-G remains unclear. Here, we report the crystal structure of a posttermination Thermus thermophilus 70S ribosome complexed with EF-G, RRF and two transfer RNAs at a resolution of 3.5 Å. The deacylated tRNA in the peptidyl (P) site moves into a previously unsuspected state of binding (peptidyl/recycling, p/R) that is analogous to that seen during initiation. The terminal end of the p/R-tRNA forms nonfavorable contacts with the 50S subunit while RRF wedges next to central inter-subunit bridges, illuminating the active roles of tRNA and RRF in dissociation of ribosomal subunits. The structure uncovers a missing snapshot of tRNA as it transits between the P and exit (E) sites, providing insights into the mechanisms of ribosome recycling and tRNA translocation.
Asunto(s)
Proteínas Bacterianas/metabolismo , ARN de Transferencia/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Thermus thermophilus/metabolismo , Proteínas Bacterianas/química , Cristalografía por Rayos X , Modelos Moleculares , Factor G de Elongación Peptídica/química , Factor G de Elongación Peptídica/metabolismo , Conformación Proteica , ARN de Transferencia/química , Proteínas Ribosómicas/química , Subunidades Ribosómicas Grandes Bacterianas/química , Subunidades Ribosómicas Grandes Bacterianas/metabolismo , Ribosomas/química , Thermus thermophilus/químicaRESUMEN
Genetic information is translated into proteins by the ribosome. Structural studies of the ribosome and of its complexes with factors and inhibitors have provided invaluable information on the mechanism of protein synthesis. Ribosome inhibitors are among the most successful antimicrobial drugs and constitute more than half of all medicines used to treat infections. However, bacterial infections are becoming increasingly difficult to treat because the microbes have developed resistance to the most effective antibiotics, creating a major public health care threat. This has spurred a renewed interest in structure-function studies of protein synthesis inhibitors, and in few cases, compounds have been developed into potent therapeutic agents against drug-resistant pathogens. In this review, we describe the modes of action of many ribosome-targeting antibiotics, highlight the major resistance mechanisms developed by pathogenic bacteria, and discuss recent advances in structure-assisted design of new molecules.
Asunto(s)
Antibacterianos/farmacología , Ribosomas/efectos de los fármacos , Animales , Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Sitios de Unión , Diseño de Fármacos , Farmacorresistencia Microbiana , Humanos , Modelos Biológicos , Modelos Moleculares , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/química , Inhibidores de la Síntesis de la Proteína/farmacología , Ribosomas/química , Ribosomas/metabolismo , Relación Estructura-ActividadRESUMEN
During translation, a plethora of protein factors bind to the ribosome and regulate protein synthesis. Many of those factors are guanosine triphosphatases (GTPases), proteins that catalyze the hydrolysis of guanosine 5'-triphosphate (GTP) to promote conformational changes. Despite numerous studies, the function of elongation factor 4 (EF-4/LepA), a highly conserved translational GTPase, has remained elusive. Here, we present the crystal structure at 2.6-Å resolution of the Thermus thermophilus 70S ribosome bound to EF-4 with a nonhydrolyzable GTP analog and A-, P-, and E-site tRNAs. The structure reveals the interactions of EF-4 with the A-site tRNA, including contacts between the C-terminal domain (CTD) of EF-4 and the acceptor helical stem of the tRNA. Remarkably, EF-4 induces a distortion of the A-site tRNA, allowing it to interact simultaneously with EF-4 and the decoding center of the ribosome. The structure provides insights into the tRNA-remodeling function of EF-4 on the ribosome and suggests that the displacement of the CCA-end of the A-site tRNA away from the peptidyl transferase center (PTC) is functionally significant.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestructura , Factores de Iniciación de Péptidos/química , Factores de Iniciación de Péptidos/ultraestructura , ARN Bacteriano/química , ARN Bacteriano/ultraestructura , ARN de Transferencia/química , ARN de Transferencia/ultraestructura , Sitios de Unión , Simulación por Computador , Simulación del Acoplamiento Molecular , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Motivos de Unión al ARN , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/ultraestructura , RibosomasRESUMEN
With bacterial resistance becoming a serious threat to global public health, antimicrobial peptides (AMPs) have become a promising area of focus in antibiotic research. AMPs are derived from a diverse range of species, from prokaryotes to humans, with a mechanism of action that often involves disruption of the bacterial cell membrane. Proline-rich antimicrobial peptides (PrAMPs) are instead actively transported inside the bacterial cell where they bind and inactivate specific targets. Recently, it was reported that some PrAMPs, such as Bac71 -35, oncocins and apidaecins, bind and inactivate the bacterial ribosome. Here we report the crystal structures of Bac71 -35, Pyrrhocoricin, Metalnikowin and two oncocin derivatives, bound to the Thermus thermophilus 70S ribosome. Each of the PrAMPs blocks the peptide exit tunnel of the ribosome by simultaneously occupying three well characterized antibiotic-binding sites and interferes with the initiation step of translation, thereby revealing a common mechanism of action used by these PrAMPs to inactivate protein synthesis. Our study expands the repertoire of PrAMPs and provides a framework for designing new-generation therapeutics.
Asunto(s)
Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Proteínas de Insectos/química , Péptidos Cíclicos/química , Biosíntesis de Proteínas/efectos de los fármacos , Ribosomas/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Sitios de Unión , Bovinos , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Insectos/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos Cíclicos/farmacología , Unión Proteica , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Ribosomas/química , Ribosomas/metabolismo , Especificidad de la Especie , Thermus thermophilus/químicaRESUMEN
Antibiotic-resistant bacteria are a global health issue necessitating the development of new effective therapeutics. Proline-rich antimicrobial peptides (PrAMPs), which include oncocins, are an extensively studied class of AMPs that counteract bacterial infection at submicromolar concentrations. Oncocins enter and kill bacteria by inhibiting certain targets rather than by acting through membrane lysis. Although they have recently been reported to bind DnaK and the bacterial ribosome, their mode of inhibition has remained elusive. Here we report the crystal structure of the oncocin derivative Onc112 bound to the Thermus thermophilus 70S ribosome. Strikingly, this 19-residue proline-rich peptide manifests the features of several known classes of ribosome inhibitors by simultaneously blocking the peptidyl transferase center and the peptide-exit tunnel of the ribosome. This high-resolution structure thus reveals the mechanism by which oncocins inhibit protein synthesis, providing an opportunity for structure-based design of new-generation therapeutics.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Inhibidores de la Síntesis de la Proteína/química , Inhibidores de la Síntesis de la Proteína/farmacología , Ribosomas/química , Péptidos Catiónicos Antimicrobianos/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Conformación Proteica , Inhibidores de la Síntesis de la Proteína/metabolismo , Ribosomas/metabolismo , Thermus thermophilus/química , Thermus thermophilus/metabolismoRESUMEN
The universally conserved GTPase elongation factor G (EF-G) catalyzes the translocation of tRNA and mRNA on the ribosome after peptide bond formation. Despite numerous studies suggesting that EF-G undergoes extensive conformational rearrangements during translocation, high-resolution structures exist for essentially only one conformation of EF-G in complex with the ribosome. Here, we report four atomic-resolution crystal structures of EF-G bound to the ribosome programmed in the pre- and posttranslocational states and to the ribosome trapped by the antibiotic dityromycin. We observe a previously unseen conformation of EF-G in the pretranslocation complex, which is independently captured by dityromycin on the ribosome. Our structures provide insights into the conformational space that EF-G samples on the ribosome and reveal that tRNA translocation on the ribosome is facilitated by a structural transition of EF-G from a compact to an elongated conformation, which can be prevented by the antibiotic dityromycin.
Asunto(s)
Factor G de Elongación Peptídica/química , Factor G de Elongación Peptídica/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Thermus thermophilus/metabolismo , Depsipéptidos/farmacología , Escherichia coli/química , Escherichia coli/metabolismo , Modelos Moleculares , ARN de Transferencia/química , Proteínas Ribosómicas/metabolismo , Ribosomas/química , Thermus thermophilus/química , Difracción de Rayos XRESUMEN
Elongation factor 4 (EF4/LepA) is a highly conserved guanosine triphosphatase translation factor. It was shown to promote back-translocation of tRNAs on posttranslocational ribosome complexes and to compete with elongation factor G for interaction with pretranslocational ribosomes, inhibiting the elongation phase of protein synthesis. Here, we report a crystal structure of EF4-guanosine diphosphate bound to the Thermus thermophilus ribosome with a P-site tRNA at 2.9 angstroms resolution. The C-terminal domain of EF4 reaches into the peptidyl transferase center and interacts with the acceptor stem of the peptidyl-tRNA in the P site. The ribosome is in an unusual state of ratcheting with the 30S subunit rotated clockwise relative to the 50S subunit, resulting in a remodeled decoding center. The structure is consistent with EF4 functioning either as a back-translocase or a ribosome sequester.
Asunto(s)
Proteínas de Escherichia coli/química , Subunidades Ribosómicas Pequeñas Bacterianas/química , Factores de Elongación Transcripcional/química , Cristalografía por Rayos X , Conformación de Ácido Nucleico , Factores de Iniciación de Péptidos , Estructura Terciaria de Proteína , ARN de Transferencia/química , Thermus thermophilusRESUMEN
In bacteria, the hybrid transfer-messenger RNA (tmRNA) rescues ribosomes stalled on defective messenger RNAs (mRNAs). However, certain gram-negative bacteria have evolved proteins that are capable of rescuing stalled ribosomes in a tmRNA-independent manner. Here, we report a 3.2 angstrom-resolution crystal structure of the rescue factor YaeJ bound to the Thermus thermophilus 70S ribosome in complex with the initiator tRNA(i)(fMet) and a short mRNA. The structure reveals that the C-terminal tail of YaeJ functions as a sensor to discriminate between stalled and actively translating ribosomes by binding in the mRNA entry channel downstream of the A site between the head and shoulder of the 30S subunit. This allows the N-terminal globular domain to sample different conformations, so that its conserved GGQ motif is optimally positioned to catalyze the hydrolysis of peptidyl-tRNA. This structure gives insights into the mechanism of YaeJ function and provides a basis for understanding how it rescues stalled ribosomes.
Asunto(s)
Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Ribosomas/química , Thermus thermophilus/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN Ribosómico/química , ARN Ribosómico/metabolismo , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/metabolismo , ARN de Transferencia de Metionina/química , ARN de Transferencia de Metionina/metabolismo , Subunidades Ribosómicas Grandes Bacterianas/química , Subunidades Ribosómicas Grandes Bacterianas/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/química , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismo , Ribosomas/metabolismo , Thermus thermophilus/metabolismo , Thermus thermophilus/ultraestructuraRESUMEN
To understand how the nucleotide sequence of ribosomal RNA determines its tertiary structure, we developed a new approach for identification of those features of rRNA sequence that are responsible for formation of different short- and long-range interactions. The approach is based on the co-analysis of several examples of a particular recurrent RNA motif. For different cases of the motif, we design combinatorial gene libraries in which equivalent nucleotide positions are randomized. Through in vivo expression of the designed libraries we select those variants that provide for functional ribosomes. Then, analysis of the nucleotide sequences of the selected clones would allow us to determine the sequence constraints imposed on each case of the motif. The constraints shared by all cases are interpreted as providing for the integrity of the motif, while those ones specific for individual cases would enable the motif to fit into the particular structural context. Here we demonstrate the validity of this approach for three examples of the so-called along-groove packing motif found in different parts of ribosomal RNA.
Asunto(s)
ARN Ribosómico/química , Proteínas Ribosómicas/química , Análisis de Secuencia de ARN , Emparejamiento Base , Biblioteca de Genes , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación de Ácido NucleicoRESUMEN
Here, we present a new recurrent RNA arrangement, the so-called adenosine wedge (A-wedge), which is found in three places of the ribosomal RNA in both ribosomal subunits. The arrangement has a hierarchical structure, consisting of elements previously described as recurrent motifs, namely, the along-groove packing motif, the A-minor and the hook-turn. Within the A-wedge, these elements are involved in different types of cause-effect relationships, providing together for the particular tertiary structure of the motif.
Asunto(s)
Adenosina/química , ARN Ribosómico/química , Secuencia de Bases , Escherichia coli/química , Escherichia coli/genética , Modelos Moleculares , Estructura Molecular , Conformación de Ácido Nucleico , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Ribosómico/genéticaRESUMEN
The along-groove packing motif is a quasi-reciprocal arrangement of two RNA double helices in which a backbone of each helix is closely packed within the minor groove of the other helix. At the center of the inter-helix contact, a GU base pair in one helix packs against a Watson-Crick base pair in the other helix. Here, based on in vivo selection from a combinatorial gene library of 16 S rRNA and on functional characterization of the selected clones, we demonstrate that the normal ribosome performance requires that helices 3 and 12 be closely packed. In some clones the Watson-Crick and GU base pairs exchange in their positions between the two helices, which affects neither the quality of the helix packing, nor the ribosome function. On the other hand, perturbations in the close packing usually lead to a substantial drop in the ribosome activity. The functionality of the clones containing such perturbations may depend on the presence of particular elements in the vicinity of the area of contact between helices 3 and 12. Such cases do not exist in natural 16 S rRNA, and their selection enriches our knowledge of the constraints imposed on the structure of ribosomal RNA in functional ribosomes.